Compositions and methods for inhibiting expression of the lect2 gene

ABSTRACT

The invention relates to antisense polynucleotide agents targeting the LECT2 gene, and methods of using such anti sense polynucleotide agents to inhibit expression of LECT2 and to treat subjects having a LECT2-associated disease, e.g., amyloidosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/564,288, filed Oct. 4, 2017, which is a U.S. National StageApplication under 35 U.S.C. § 371 of International Application No.PCT/US2016/026676, filed Apr. 8, 2016, which claims the benefit of U.S.Provisional Application No. 62/144,695, filed Apr. 8, 2015, the contentsof which are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on April 6, 2016, isnamed A2038-7221WO_SL.txt and is 96,680 bytes in size.

FIELD OF THE INVENTION

The invention relates to the specific inhibition of the expression ofthe LECT2 gene.

BACKGROUND OF THE INVENTION

Amyloidosis is a group of diseases characterized by deposition ofinsoluble fibrous protein aggregates, called amyloids, in organs ortissues. Amyloids can form from mutant or wild type proteins. One systemof nomenclature for amyloid diseases uses an abbreviation for theprotein that forms amyloid deposits, preceded by the letter “A.” Thus,for example, ALECT2 is the abbreviation for an amyloidosis involvingdeposit of amyloids formed from leukocyte cell derived chemotacticfactor-2 (ALECT2).

LECT2 amyloidosis (ALECT2) is one of the most recently discovered typesof amyloidosis. LECT2 amyloidosis has been observed in individuals withrenal or hepatic amyloidosis. This form of amyloidosis can present withnephrotic syndrome or with liver involvement (e.g., hepatitis, e.g.,chronic hepatitis). It may be particularly prevalent in MexicanAmericans and/or individuals who are homozygous for the G alleleencoding valine at position 40 in the mature LECT2 protein (or atposition 58 in the unprocessed protein). Treatments for LECT2amyloidosis are limited, and new treatments are needed.

SUMMARY OF THE INVENTION

The present invention provides antisense polynucleotide agents andcompositions comprising such agents which target nucleic acids encodinga LECT2 gene and interfere with the normal function of the targetednucleic acid. The LECT2 nucleic acid may be within a cell, e.g., a cellwithin a subject, such as a human. The present invention also providesmethods and combination therapies for treating a subject having adisorder that would benefit from inhibiting or reducing the expressionof a LECT2 mRNA, e.g., a LECT2-associated disease, such as amyloidosis,e.g., a LECT2 amyloidosis (ALECT2) using the antisense polynucleotideagents and compositions of the invention.

Accordingly, in one aspect, the present invention provides antisensepolynucleotide agents for inhibiting expression of a LECT2 gene. Theagents comprise about 4 to about 50 contiguous nucleotides, wherein thenucleotide sequence of the agent is about 80% complementary over itsentire length to the equivalent region of the nucleotide sequence of anyone of SEQ ID NOs: 1-4.

In one embodiment, the equivalent region is one of the target regions ofSEQ ID NO: 1 provided in Table 3, e.g., SEQ ID NOs: 109-204.

In one embodiment, the nucleotides of an antisense polynucleotide agentof the invention are un-modified, and do not comprise, e.g., chemicalmodifications and/or conjugations known in the art and described herein(e.g., as shown in Table 3). In another embodiment, one or more (e.g.,at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) of thenucleotides of an antisense polynucleotide agent of the invention ischemically modified (e.g., as shown in Table 3).

In another aspect, the present invention provides antisensepolynucleotide agents for inhibiting expression of a LECT2 gene, whereinthe agent comprises at least 8 contiguous nucleotides differing by nomore than 3 nucleotides from any one of the nucleotide sequences listedin Table 3.

In some embodiments, substantially all of the nucleotides of theantisense polynucleotide agents of the invention are modifiednucleotides. In other embodiment, all of the nucleotides of theantisense polynucleotide agent are modified nucleotides.

The antisense polynucleotide agent may be 10 to 40 nucleotides inlength; 10 to 30 nucleotides in length; 18 to 30 nucleotides in length;10 to 24 nucleotides in length; 18 to 24 nucleotides in length; 14-20nucleotides in length; or 14 or 20 nucleotides in length.

In one embodiment, the modified nucleotide comprises a modified sugarmoiety selected from the group consisting of: a 2′-O-methoxyethylmodified sugar moiety, a 2′-methoxy modified sugar moiety, a 2′-O-alkylmodified sugar moiety, and a bicyclic sugar moiety.

In one embodiment, the bicyclic sugar moiety has a (—CH2—)n groupforming a bridge between the 2′ oxygen and the 4′ carbon atoms of thesugar ring, wherein n is 1 or 2.

In another embodiment, the modified nucleotide is a 5-methylcytosine.

In one embodiment, the modified nucleotide comprises a modifiedinternucleoside linkage, such as a phosphorothioate internucleosidelinkage.

In one embodiment, an agent of the invention comprises a plurality of2′-deoxynucleotides flanked on each side by at least one nucleotidehaving a modified sugar moiety.

In one embodiment, the agent is a gapmer comprising a gap segmentcomprised of linked 2′-deoxynucleotides positioned between a 5′ and a 3′wing segment.

In one embodiment, the modified sugar moiety is selected from the groupconsisting of a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxymodified sugar moiety, a 2′-O-alkyl modified sugar moiety, and abicyclic sugar moiety.

In one embodiment, the 5′-wing segment is 1 to 6 nucleotides in length,e.g., 2, 3, 4, or 5 nucleotides in length.

In one embodiment, the 3′-wing segment is 1 to 6 nucleotides in length,e.g., 2, 3, 4, or 5 nucleotides in length.

In one embodiment, the gap segment is 5 to 14 nucleotides in length,e.g., 10 nucleotides in length.

In one aspect, the present invention provides antisense polynucleotideagent for inhibiting LECT2 gene expression, comprising a gap segmentconsisting of linked deoxynucleotides; a 5′-wing segment consisting oflinked nucleotides; a 3′-wing segment consisting of linked nucleotides;wherein the gap segment is positioned between the 5′-wing segment andthe 3′-wing segment and wherein each nucleotide of each wing segmentcomprises a modified sugar.

In one embodiment, the gap segment is ten 2′-deoxynucleotides in lengthand each of the wing segments is five nucleotides in length.

In another embodiment, the gap segment is ten 2′-deoxynucleotides inlength and each of the wing segments is four nucleotides in length.

In yet another embodiment, the gap segment is ten 2′-deoxynucleotides inlength and each of the wing segments is three nucleotides in length.

In another embodiment, the gap segment is ten 2′-deoxynucleotides inlength and each of the wing segments is two nucleotides in length.

In one embodiment, the modified sugar moiety is selected from the groupconsisting of a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxymodified sugar moiety, a 2′-O-alkyl modified sugar moiety, and abicyclic sugar moiety.

In some embodiments, the agents of the invention further comprise aligand.

In one embodiment, the agent is conjugated to the ligand at the3′-terminus.

In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc)derivative.

In one embodiment, the ligand is

In one embodiment, the antisense polynucleotide agent has a region thatis substantially complementary to a portion of a LECT2 mRNA, e.g., ahuman LECT2 mRNA (e.g., a human LECT2 mRNA as provided in NM_002302.2(SEQ ID NO: 1).

In embodiments, the antisense polynucleotide agent has a region that issubstantially complementary to a portion of a LECT2 mRNA transcript thathas a A to G substitution at nucleotide position 373 of SEQ ID NO: 1. Inembodiments, the mRNA transcript encodes valine at position 40 in themature LECT2 protein (or amino acid 58 in the unprocessed protein). Inembodiments, the mRNA transcript encodes isoleucine at position 40 inthe mature LECT2 protein (or amino acid 58 in the unprocessed protein).

In one embodiment, an antisense polynucleotide agent as described hereintargets a wildtype LECT2 RNA transcript variant, and in anotherembodiment, the antisense polynucleotide agent targets a mutanttranscript (e.g., a LECT2 RNA carrying an allelic variant). For example,an anti sense polynucleotide agent featured in the invention can targeta polymorphic variant, such as a single nucleotide polymorphism (SNP),of LECT2.

In one aspect, the present invention provides pharmaceuticalcompositions for inhibiting expression of a LECT2 gene comprising theagents of the invention.

In one embodiment, the agent is present in an unbuffered solution, suchas saline or water.

In another embodiment, the agent is present in a buffer solution, suchas a buffer comprising acetate, citrate, prolamine, carbonate, orphosphate or any combination thereof.

In one embodiment, the buffer solution is phosphate buffered saline(PBS).

In another aspect, the present invention provides pharmaceuticalcomposition comprising an agent of the invention and a lipidformulation, such as a lipid formulation comprising an LNP or a MC3.

In one aspect, the present invention provides methods of inhibitingLECT2 gene expression in a cell. The methods include contacting the cellwith the agent of the invention or a pharmaceutical composition of theinvention; and maintaining the cell for a time sufficient to obtainantisense inhibition of a LECT2 gene, thereby inhibiting expression ofthe LECT2 gene in the cell.

In one embodiment, the cell is within a subject.

In one embodiment, the subject is a human.

In one embodiment, the LECT2 gene expression is inhibited by at leastabout 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, about 95%, about 98% or about 100%.

In another aspect, the present invention provides methods of treating asubject having a disease or disorder that would benefit from reductionin LECT2 gene expression. The methods include administering to thesubject a therapeutically effective amount of an agent of the inventionor a pharmaceutical composition of the invention, thereby treating thesubject.

In yet another aspect, the present invention provides methods ofpreventing at least one symptom in a subject having a disease ordisorder that would benefit from reduction in LECT2 gene expression. Themethods include administering to the subject a prophylacticallyeffective amount of the agent of the invention or a pharmaceuticalcomposition of the invention, thereby preventing at least one symptom inthe subject having a disorder that would benefit from reduction in LECT2gene expression.

In one embodiment, the administration of the antisense polynucleotideagent to the subject causes a decrease in amyloid deposition (e.g., bypreventing amyloid deposition or reducing amyloid deposition, e.g., byreducing size, number, or extent of amyloid deposits) or symptomsassociated with amyloid deposition and/or a decrease in LECT2 proteinlevels.

In one embodiment, the administration of the antisense polynucleotideagent to the subject inhibits amyloid deposition (e.g., by preventingamyloid deposition or reducing amyloid deposition, e.g., by reducingsize, number, or extent of amyloid deposits). The inhibition optionallyinvolves an inhibition of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50% or more compared to a reference, (e.g., a control that isuntreated or treated with a non-targeting dsRNA (e.g., a dsRNA that doesnot target LECT2)).

In one embodiment, the disorder is a LECT2-associated disease, e.g., aLECT2 amyloidosis. In some embodiments, the LECT2 amyloidosis is a renalamyloidosis. In some embodiments, the LECT2 amyloidosis involves amyloiddeposition in the kidney. In some embodiments, LECT2 amyloidosis isassociated with renal disease (e.g., nephrotic syndrome). In someembodiments, the amyloidosis is associated with proteinuria. In someembodiments, proteinuria is absent. In some embodiments, the LECT2amyloidosis is a hepatic amyloidosis. In some embodiments, the LECT2amyloidosis involves amyloid deposition in the liver. In someembodiments, the LECT2 amyloidosis is associated with inflammation inthe liver (e.g., hepatitis, e.g., chronic hepatitis).

In one embodiment the subject is human. In some embodiments, the subjectis of Mexican descent (e.g., a Mexican American).

In embodiments, the subject carries the G allele of the LECT2 gene thatencodes valine at position 40 in the mature protein (or amino acid 58 inthe unprocessed protein). In embodiments, the subject is homozygous forthe G allele (G/G genotype). In embodiments, a LECT2 protein expressedin the subject has valine at position 40 in the mature protein (or atamino acid 58 in the unprocessed protein).

In one embodiment, the agent is administered at a dose of about 0.01mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 50 mg/kg.

In one embodiment, the agent is administered at a dose of about 10 to100 mg/kg or 0.5 to 10 mg/kg.

In one embodiment, the agent is administered to the subject once a week.

In another embodiment, the agent is administered to the subject twice aweek.

In yet another embodiment, the agent is administered to the subjecttwice a month.

In one embodiment, the agent is administered to the subjectsubcutaneously.

In another embodiment, a composition the agent featured in the inventionis administered in conjunction with a second therapy for a disorderrelated to LECT2 expression (e.g., a LECT2 amyloidosis). The agent canbe administered before, after, or concurrent with a second therapy. Inembodiments, the agent is administered before the second therapy. Inembodiments, the agent is administered after the second therapy. Inembodiments, the agent is administered concurrent with the secondtherapy.

In some embodiments, the second therapy is a non-antisensepolynucleotide therapeutic agent that is effective to treat the disorderor symptoms of the disorder.

In some embodiments, the disorder to be treated by the compositions ormethods disclosed herein is a LECT2 amyloidosis that affects kidneyfunction, e.g., through amyloid deposition in the kidney. In some suchembodiments, the agent is administered in conjunction with a therapythat supports kidney function (e.g., dialysis). In embodiments, theagent is administered in conjunction with a diuretic, an ACE(angiotensin converting enzyme) inhibitor, an angiotensin receptorblocker, and/or dialysis, e.g., to support or manage kidney function.

In some embodiments, the disorder to be treated by the compositions ormethods disclosed herein is a LECT2 amyloidosis involving amyloiddeposits in the liver. In some such embodiments, the agent isadministered in conjunction with a therapy that supports liver function.

In some embodiments, the disorder to be treated by the compositions ormethods disclosed herein is a LECT2 amyloidosis, and the agent isadministered in conjunction with removal of all or part of the organ(s)affected by the amyloidosis (e.g., resection of all or part of kidney orliver tissue affected by the amyloidosis). The removal is optionallyconducted in conjunction with a replacement of all or part of the organremoved (e.g., in conjunction with a kidney or liver organ transplant).

In one embodiment, the methods of the invention further includeadministering an anti-LECT2 antibody, or antigen-binding fragmentthereof, to the subject.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides antisense polynucleotide agents andcompositions comprising such agents which target nucleic acids encodingLECT2 (e.g., mRNA encoding LECT2 as provided in, for example, any one ofSEQ ID NOs: 1-4). The antisense polynucleotide agents bind to nucleicacids encoding LECT2 via, e.g., Watson-Crick base pairing, and interferewith the normal function of the targeted nucleic acid.

The antisense polynucleotide agents of the invention include anucleotide sequence which is about 4 to about 50 nucleotides or less inlength and which is about 80% complementary to at least part of an mRNAtranscript of a LECT2 gene. The use of these antisense polynucleotideagents enables the targeted inhibition of RNA expression and/or activityof a LECT2 gene in mammals.

The present inventors have demonstrated that antisense polynucleotideagents targeting LECT2 can mediate antisense inhibition in vitroresulting in significant inhibition of expression of a LECT2 gene. Thus,methods and compositions including these antisense polynucleotide agentsare useful for treating a subject who would benefit by a reduction inthe levels and/or activity of a LECT2 protein, such as a subject havinga LECT2 -associated disease, such as e.g., amyloidosis, e.g., LECT2amyloidosis.

The present invention also provides methods and combination therapiesfor treating a subject having a disorder that would benefit frominhibiting or reducing the expression of a LECT2 gene, e.g., a LECT2-associated disease, such as amyloidosis, e.g. a LECT2 amyloidosis(ALECT2) using the antisense polynucleotide agents and compositions ofthe invention.

The present invention also provides methods for preventing at least onesymptom, e.g., amyloid deposition, in a subject having a disorder thatwould benefit from inhibiting or reducing the expression of a LECT2gene, e.g., a LECT2 -associated disease, such as amyloidosis, e.g. aLECT2 amyloidosis (ALECT2). The present invention further providescompositions comprising anti sense polynucleotide agents which effectantisense inhibition of a LECT2 gene. The LECT2 gene may be within acell, e.g., a cell within a subject, such as a human.

The following detailed description discloses how to make and useantisense polynucleotide agents to inhibit the mRNA and/or proteinexpression of a LECT2 gene, as well as compositions, uses, and methodsfor treating subjects having diseases and disorders that would benefitfrom inhibition and/or reduction of the expression of this gene.

I. Definitions

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

As used herein, “LECT2” refers to leukocyte chemotactic factor 2 (alsoknown as leukocyte cell-derived chemotaxin 2, chondromodulin-II, chm-IIor chm2). See, e.g., Yamagoe S et al. Genomics, 1998 Mar. 15;48(3):324-9. LECT2 was first identified as a novel neutrophilchemotactic protein and is identical with chondromodulin II, a growthstimulator for chondrocytes and osteoblasts. The human LECT2 gene wasmapped to chromosome 5q31.1-q32. Ibid.

The sequence of a human LECT2 mRNA transcript can be found atNM_002302.2 (SEQ ID NO: 1). The sequence of a macaque LECT2 mRNA can befound at NM_001194884.2 (SEQ ID NO: 2). The sequence of a mouse LECT2mRNA can be found at NM_010702.1 and at NM_010702.2 (SEQ ID NO: 3), andthe sequence of a rat LECT2 mRNA can be found at NM_001108405.1 (SEQ IDNO: 4).

The human LECT2 protein is a secreted, 16 kDa protein. The LECT2 proteinis secreted by the liver. It has high sequence similarity to thechondromodulin repeat regions of the chicken myb-induced myeloid 1protein. Polymorphism in the LECT2 gene has been associated withrheumatoid arthritis. Ibid.

LECT2 is expressed in various tissues, including the brain and stomachas well as the liver. Koshimizu, Y & Ohtomi, M. (2010) Brain Res.1311:1-11. In a study using indirect immunoperoxidase staining toinvestigate the expression of LECT2 in normal and diseased human organsand tissues other than liver, it was found that LECT2 was generallyexpressed in vascular, endothelial and smooth muscle cells, adipocytes,cerebral nerve cells, apical squamous epithelia, parathyroid cells,sweat and sebaceous glandular epithelia, Hassall bodies and somemononuclear cells in immunohematopoeietic tissue. This protein wasgenerally negative, although occasionally positively stained inosteoblasts, chondrocytes, cardiac and skeletal muscle cells, smoothmucle cells of the gastrointestinal tract, and the epithelial cells ofsome tissues. Nagai et al. (1998) Pathol 48(11):882-6.

The human LECT2 gene codes for 151 amino acids including an 18 aminoacid signal peptide. The secreted protein has 133 residues. A G/Apolymorphism at nucleotide 172 in exon 3 of the gene (codon change GTCto ATC) has been identified and accounts for the presence of eithervaline or isoleucine at position 58 of the unprocessed protein (orposition 40 of the mature protein). The G allele has an overallfrequency of 0.477 and a frequency range of 0.6-0.7 in individuals ofEuropean descent. See Benson, M. D. et al. (2008) Kidney International,74: 218-222; Murphy, C. L. et al. (2010) Am J Kidney Dis,56(6):1100-1107. Patients with LECT2 amyloidosis typically arehomozygous for the G allele. Without wishing to be bound by theory, ithas been suggested that replacement of the buried isoleucine (A allele)side chain with valine (G allele) could destabilize the protein andpossibly account for the amyloidogenic propensity of this LECT2 variant.Murphy, C. L. et al. (2010) Am J Kidney Dis, 56(6):1100-1107.

As used herein, a “LECT2 amyloidosis” or “ALECT2” includes anamyloidosis involving deposits of amyloid or amyloid fibrils thatcontain a LECT2 protein (e.g., any polymorphic variant of a LECT2protein) or a portion of a LECT2 protein. The LECT2 protein can be avariant (e.g., a mutant) LECT2 protein. The amyloidosis can be systemicor local. In embodiments, the LECT2 amyloidosis involves amyloiddeposits in the kidney and/or liver.

The terms “antisense polynucleotide agent” “antisense compound”, and“agent” as used interchangeably herein, refer to an agent comprising asingle-stranded oligonucleotide that contains RNA as that term isdefined herein, and which targets nucleic acid molecules encoding LECT2(e.g., mRNA encoding LECT2 as provided in, for example, any one of SEQID NOs:1-4). The antisense polynucleotide agents specifically bind tothe target nucleic acid molecules via hydrogen bonding (e.g.,Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) andinterfere with the normal function of the targeted nucleic acid (e.g.,by an antisense mechanism of action). This interference with ormodulation of the function of a target nucleic acid by thepolynucleotide agents of the present invention is referred to as“antisense inhibition.”

The functions of the target nucleic acid molecule to be interfered withmay include functions such as, for example, translocation of the RNA tothe site of protein translation, translation of protein from the RNA,splicing of the RNA to yield one or more mRNA species, and catalyticactivity which may be engaged in or facilitated by the RNA.

In some embodiments, antisense inhibition refers to “inhibiting theexpression” of target nucleic acid levels and/or target protein levelsin a cell, e.g., a cell within a subject, such as a mammalian subject,in the presence of the antisense polynucleotide agent complementary to atarget nucleic acid as compared to target nucleic acid levels and/ortarget protein levels in the absence of the antisense polynucleotideagent. For example, the antisense polynucleotide agents of the inventioncan inhibit translation in a stoichiometric manner by base pairing tothe mRNA and physically obstructing the translation machinery, see Dias,N. et al., (2002) Mol Cancer Ther 1:347-355.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a LECT2 gene, including mRNA that is a product of RNA processing of aprimary transcription product.

As used herein, “target nucleic acid” refers to a nucleic acid moleculeto which an antisense polynucleotide agent specifically hybridizes.

As used herein, the term “specifically hybridizes” refers to anantisense polynucleotide agent having a sufficient degree ofcomplementarity between the antisense polynucleotide agent and a targetnucleic acid to induce a desired effect, while exhibiting minimal or noeffects on non-target nucleic acids under conditions in which specificbinding is desired, e.g., under physiological conditions in the case ofin vivo assays and therapeutic treatments.

A target sequence may be from about 4-50 nucleotides in length, e.g.,8-45, 10-45, 10-40, 10-35, 10-30, 10-20, 11-45, 11-40, 11-35, 11-30,11-20, 12-45, 12-40, 12-35, 12-30, 12-25, 12-20, 13-45, 13-40, 13-35,13-30, 13-25, 13-20, 14-45, 14-40, 14-35, 14-30, 14-25, 14-20, 15-45,15-40, 15-35, 15-30, 15-25, 15-20, 16-45, 16-40, 16-35, 16-30, 16-25,16-20, 17-45, 17-40, 17-35, 17-30, 17-25, 17-20, 18-45, 18-40, 18-35,18-30, 18-25, 18-20, 19-45, 19-40, 19-35, 19-30, 19-25, 19-20, e.g., 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, or 50 contiguous nucleotides of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a LECT2 gene. Ranges and lengths intermediate to the above recitedranges and lengths are also contemplated to be part of the invention.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” are used herein with respect to the base matching betweenan antisense polynucleotide agent and a target sequence. Theterm“complementarity” refers to the capacity for pairing betweennucleobases of a first nucleic acid and a second nucleic acid.

As used herein, an antisense polynucleotide agent that is “substantiallycomplementary to at least part of” a messenger RNA (mRNA) refers to anantisense polynucleotide agent that is substantially complementary to acontiguous portion of the mRNA of interest (e.g., an mRNA encodingLECT2). For example, a polynucleotide is complementary to at least apart of a LECT2 mRNA if the sequence is substantially complementary to anon-interrupted portion of an mRNA encoding LECT2.

As used herein, the term “region of complementarity” refers to theregion of the antisense polynucleotide agent that is substantiallycomplementary to a sequence, for example a target sequence, e.g., aLECT2 nucleotide sequence, as defined herein. Where the region ofcomplementarity is not fully complementary to the target sequence, themismatches can be in the internal or terminal regions of the molecule.Generally, the most tolerated mismatches are in the terminal regions,e.g., within 5, 4, 3, or 2 nucleotides of the 5′- and/or 3′-terminus ofthe antisense polynucleotide.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of apolynucleotide comprising the first nucleotide sequence to hybridize andform a duplex structure under certain conditions with the secondnucleotide sequence, as will be understood by the skilled person. Suchconditions can, for example, be stringent conditions, where stringentconditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C. or 70° C. for 12-16 hours followed by washing (see, e.g., “MolecularCloning: A Laboratory Manual,” Sambrook, et al. (1989) Cold SpringHarbor Laboratory Press). Other conditions, such as physiologicallyrelevant conditions as can be encountered inside an organism, can apply.The skilled person will be able to determine the set of conditions mostappropriate for a test of complementarity of two sequences in accordancewith the ultimate application of the nucleotides.

Complementary sequences include those nucleotide sequences of anantisense polynucleotide agent of the invention that base-pair to asecond nucleotide sequence over the entire length of one or bothnucleotide sequences. Such sequences can be referred to as “fullycomplementary” with respect to each other herein. However, where a firstsequence is referred to as “substantially complementary” with respect toa second sequence herein, the two sequences can be fully complementary,or they can form one or more, but generally not more than 5, 4, 3 or 2mismatched base pairs upon hybridization for a duplex up to 30 basepairs, while retaining the ability to hybridize under the conditionsmost relevant to their ultimate application, e.g., antisense inhibitionof target gene expression.

“Complementary” sequences, as used herein, can also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in so far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogstein base pairing.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively. However, it will be understood that the terms“deoxyribonucleotide”, “ribonucleotide” and “nucleotide” can also referto a modified nucleotide, as further detailed below, or a surrogatereplacement moiety (see, e.g., Table 2). The skilled person is wellaware that guanine, cytosine, adenine, and uracil can be replaced byother moieties without substantially altering the base pairingproperties of an oligonucleotide comprising a nucleotide bearing suchreplacement moiety. For example, without limitation, a nucleotidecomprising inosine as its base can base pair with nucleotides containingadenine, cytosine, or uracil. Hence, nucleotides containing uracil,guanine, or adenine can be replaced in the nucleotide sequences of theagents featured in the invention by a nucleotide containing, forexample, inosine. In another example, adenine and cytosine anywhere inthe oligonucleotide can be replaced with guanine and uracil,respectively to form G-U Wobble base pairing with the target mRNA.Sequences containing such replacement moieties are suitable for thecompositions and methods featured in the invention.

A “nucleoside” is a base-sugar combination. The “nucleobase” (also knownas “base”) portion of the nucleoside is normally a heterocyclic basemoiety. “Nucleotides” are nucleosides that further include a phosphategroup covalently linked to the sugar portion of the nucleoside. Forthose nucleosides that include a pentofuranosyl sugar, the phosphategroup can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar.“Polynucleotides,” also referred to as “oligonucleotides,” are formedthrough the covalent linkage of adjacent nucleosides to one another, toform a linear polymeric oligonucleotide. Within the polynucleotidestructure, the phosphate groups are commonly referred to as forming theinternucleoside linkages of the polynucleotide.

In general, the majority of nucleotides of the antisense polynucleotideagents are ribonucleotides, but as described in detail herein, theagents may also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide. In addition, as used in this specification, an“antisense polynucleotide agent” may include nucleotides (e.g.,ribonucleotides or deoxyribonucleotides) with chemical modifications; anantisense polynucleotide agent may include substantial modifications atmultiple nucleotides.

As used herein, the term “modified nucleotide” refers to a nucleotidehaving, independently, a modified sugar moiety, a modifiedinternucleotide linkage, and/or modified nucleobase. Thus, the termmodified nucleotide encompasses substitutions, additions or removal of,e.g., a functional group or atom, to internucleoside linkages, sugarmoieties, or nucleobases. The modifications suitable for use in theantisense polynucleiotde agents of the invention include all types ofmodifications disclosed herein or known in the art. Any suchmodifications, as used in nucleotides, are encompassed by “antisensepolynucleotide agent” for the purposes of this specification and claims.

As used herein, a “subject” is an animal, such as a mammal, including aprimate (such as a human, a non-human primate, e.g., a monkey, and achimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, ahorse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog,a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or agoose). In an embodiment, the subject is a human, such as a human beingtreated or assessed for a disease, disorder or condition that wouldbenefit from reduction in LECT2 expression; a human at risk for adisease, disorder or condition that would benefit from reduction inLECT2 expression; a human having a disease, disorder or condition thatwould benefit from reduction in LECT2 expression; and/or human beingtreated for a disease, disorder or condition that would benefit fromreduction in LECT2 expression as described herein.

As used herein, the terms “treating” or “treatment” refer to abeneficial or desired result including, but not limited to, alleviationor amelioration of one or more symptoms associated with LECT2expression; or diminishing the extent of LECT2 expression. For example,the methods featured herein, when employed to treat a LECT2 amyloidosis,may serve to inhibit amyloid deposition, to reduce or prevent one ormore symptoms of the amyloidosis, or to reduce the risk or severity ofassociated conditions.

The term “lower” in the context of the level of LECT2 in a subject or adisease marker or symptom refers to a statistically significant decreasein such level. The decrease can be, for example, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or more and is preferably down to a level accepted aswithin the range of normal for an individual without such disorder.

As used herein, “prevention” or “preventing,” when used in reference toa disease, disorder or condition thereof, that would benefit from areduction in expression of a LECT2 gene, refers to a reduction in thelikelihood that a subject will develop a symptom associated with such adisease, disorder, or condition, e.g., a symptom of unwanted LECT2expression, such as amyloid deposition.

II. Antisense Polynucleotide Agents of the Invention

The present invention provides antisense polynucleotide agents, andcompositions comprising such agents, which target a LECT2 gene andinhibit the expression of the LECT2 gene. In one embodiment, theantisense polynucleotide agents inhibit the expression of a LECT2 genein a cell, such as a cell within a subject, e.g., a mammal, such as ahuman having a LECT2-associated disease, e.g., amyloidosis, e.g. a LECT2amyloidosis (ALECT2).

The antisense polynucleotide agents of the invention include a region ofcomplementarity which is complementary to at least a part of an mRNAformed in the expression of a LECT2 gene. The region of complementaritymay be about 50 nucleotides or less in length (e.g., about 50, 49, 48,47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30,29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, or 4 nucleotides or less in length). Upon contactwith a cell expressing the LECT2 gene, the antisense polynucleotideagent inhibits the expression of the LECT2 gene (e.g., a human, aprimate, a non-primate, or a bird LECT2 gene) by at least about 10% asassayed by, for example, a PCR or branched DNA (bDNA)-based method, orby a protein-based method, such as by immunofluorescence analysis,using, for example, Western Blotting or flow cytometric techniques.

The region of complementarity between an antisense polynucleotide agentand a target sequence may be substantially complementary (e.g., there isa sufficient degree of complementarity between the antisensepolynucleotide agent and a target nucleic acid to so that theyspecifically hybridize and induce a desired effect), but is generallyfully complementary to the target sequence. The target sequence can bederived from the sequence of an mRNA formed during the expression of aLECT2 gene.

Accordingly, in one aspect, an antisense polynucleotide agent of theinvention specifically hybridizes to a target nucleic acid molecule,such as the mRNA encoding LECT2, and comprises a contiguous nucleotidesequence which corresponds to the reverse complement of a nucleotidesequence of any one of SEQ ID NOs: 1-4, or a fragment of any one of SEQID NOs: 1-4.

In some embodiments, the antisense polynucleotide agents of theinvention may be substantially complementary to the target sequence. Forexample, an antisense polynucleotide agent that is substantiallycomplementary to the target sequence may include a contiguous nucleotidesequence comprising no more than 5 mismatches (e.g., no more than 1, nomore than 2, no more than 3, no more than 4, or no more than 5mismatches) when hybridizing to a target sequence, such as to thecorresponding region of a nucleic acid which encodes a mammalian LECT2mRNA. In some embodiments, the contiguous nucleotide sequence comprisesno more than a single mismatch when hybridizing to the target sequence,such as the corresponding region of a nucleic acid which encodes amammalian LECT2 mRNA.

In some embodiments, the antisense polynucleotide agents of theinvention that are substantially complementary to the target sequencecomprise a contiguous nucleotide sequence which is at least about 80%complementary over its entire length to the equivalent region of thenucleotide sequence of any one of SEQ ID NOs: 1-4, or a fragment of anyone of SEQ ID NOs: 1-4, such as about 85%, about 86%, about 87%, about88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In some embodiments, an antisense polynucleotide agent comprises acontiguous nucleotide sequence which is fully complementary over itsentire length to the equivalent region of the nucleotide sequence of anyone of SEQ ID NOs: 1-4 (or a fragment of any one of SEQ ID

NOs: 1-4). For example, the nucleotide sequence of Oligo: A-133895.1 isfully complementary over its entire length to the equivalent region ofnucleotides 12-31 of NM_002302.2 (SEQ ID NO: 1) (see, e.g., Table 3).Similarly, the nucleotide sequence of Sequence ID NO: A-133925.1 isfully complementary over its entire length to the equivalent region ofnucleotides 561-581 of NM 002302.2 (SEQ ID NO: 1) (see, e.g., Table 3)and the nucleotide sequence of Sequence ID

NO: A-133953.1 is fully complementary over its entire length to theequivalent region of nucleotides 870-889 of NM_002302.2 (SEQ ID NO: 1)(see, e.g., Table 3).

An antisense polynucleotide agent may comprise a contiguous nucleotidesequence of about 4 to about 50 nucleotides in length, e.g., 8-49, 8-48,8-47, 8-46, 8-45, 8-44, 8-43, 8-42, 8-41, 8-40, 8-39, 8-38, 8-37, 8-36,8-35, 8-34, 8-33, 8-32, 8-31, 8-30, 8-29, 8-28, 8-27, 8-26, 8-25, 8-24,8-23, 8-22, 8-21, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12,8-11, 8-10, 8-9, 10-49, 10-48, 10-47, 10-46, 10-45, 10-44, 10-43, 10-42,10-41, 10-40, 10-39, 10-38, 10-37, 10-36, 10-35, 10-34, 10-33, 10-32,10-31, 10-30, 10-29, 10-28, 10-27, 10-26, 10-25, 10-24, 10-23, 10-22,10-21, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12,10-11,11-49, 11-48, 11-47, 11-46, 11-45, 11-44, 11-43, 11-42, 11-41,11-40, 11-39, 11-38, 11-37, 11-36, 11-35, 11-34, 11-33, 11-32, 11-31,11-30, 11-29, 11-28, 11-27, 11-26, 11-25, 11-24, 11-23, 11-22, 11-21,11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-49,12-48, 12-47, 12-46, 12-45, 12-44, 12-43, 12-42, 12-41, 12-40, 12-39,12-38, 12-37, 12-36, 12-35, 12-34, 12-33, 12-32, 12-31, 12-30, 12-29,12-28, 12-27, 12-26, 12-25, 12-24, 12-23, 12-22, 12-21, 12-20, 12-19,12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-49, 13-48, 13-47, 13-46,13-45, 13-44, 13-43, 13-42, 13-41, 13-40, 13-39, 13-38, 13-37, 13-36,13-35, 13-34, 13-33, 13-32, 13-31, 13-30, 13-29, 13-28, 13-27, 13-26,13-25, 13-24, 13-23, 13-22, 13-21, 13-20, 13-19, 13-18, 13-17, 13-16,13-15, 13-14, 14-49, 14-48, 14-47, 14-46, 14-45, 14-44, 14-43, 14-42,14-41, 14-40, 14-39, 14-38, 14-37, 14-36, 14-35, 14-34, 14-33, 14-32,14-31, 14-30, 14-29, 14-28, 14-27, 14-26, 14-25, 14-24, 14-23, 14-22,14-21, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-49, 15-48, 15-47,15-46, 15-45, 15-44, 15-43, 15-42, 15-41, 15-40, 15-39, 15-38, 15-37,15-36, 15-35, 15-34, 15-33, 15-32, 15-31, 15-30, 15-29, 15-28, 15-27,15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17,15-16,16-49, 16-48, 16-47, 16-46, 16-45, 16-44, 16-43, 16-42, 16-41,16-40, 16-39, 16-38, 16-37, 16-36, 16-35, 16-34, 16-33, 16-32, 16-31,16-30, 16-29, 16-28, 16-27, 16-26, 16-25, 16-24, 16-23, 16-22, 16-21,16-20, 16-19, 16-18, 16-17, 17-49, 17-48, 17-47, 17-46, 17-45, 17-44,17-43, 17-42, 17-41, 17-40, 17-39, 17-38, 17-37, 17-36, 17-35, 17-34,17-33, 17-32, 17-31, 17-30, 17-29, 17-28, 17-27, 17-26, 17-25, 17-24,17-23, 17-22, 17-21, 17-20, 17-19, 17-18, 18-49, 18-48, 18-47, 18-46,18-45, 18-44, 18-43, 18-42, 18-41, 18-40, 18-39, 18-38, 18-37, 18-36,18-35, 18-34, 18-33, 18-32, 18-31, 18-30, 18-29, 18-28, 18-27, 18-26,18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-49, 19-48, 19-47, 19-46,19-45, 19-44, 19-43, 19-42, 19-41, 19-40, 19-39, 19-38, 19-37, 19-36,19-35, 19-34, 19-33, 19-32, 19-31, 19-30, 19-29, 19-28, 19-27, 19-26,19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-49, 20-48, 20-47, 20-46,20-45, 20-44, 20-43, 20-42, 20-41, 20-40, 20-39, 20-38, 20-37, 20-36,20-35, 20-34, 20-33, 20-32, 20-31, 20-30, 20-29, 20-28, 20-27, 20-26,20-25, 20-24,20-23, 20-22, 20-21, 21-49, 21-48, 21-47, 21-46, 21-45,21-44, 21-43, 21-42, 21-41, 21-40, 21-39, 21-38, 21-37, 21-36, 21-35,21-34, 21-33, 21-32, 21-31, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25,21-24, 21-23, 21-22, 22-49, 22-48, 22-47, 22-46, 22-45, 22-44, 22-43,22-42, 22-41, 22-40, 22-39, 22-38, 22-37, 22-36, 22-35, 22-34, 22-33,22-32, 22-31, 22-30, 22-29, 22-28, 22-27, 22-26, 22-25, 22-24, 22-23,23-49, 23-48, 23-47, 23-46, 23-45, 23-44, 23-43, 23-42, 23-41, 23-40,23-39, 23-38, 23-37, 23-36, 23-35, 23-34, 23-33, 23-32, 23-31, 23-30,23-29, 23-28, 23-27, 23-26, 23-25, 23-24, 24-49, 24-48, 24-47, 24-46,24-45, 24-44, 24-43, 24-42, 24-41, 24-40, 24-39, 24-38, 24-37, 24-36,24-35, 24-34, 24-33, 24-32, 24-31, 24-30, 24-29, 24-28, 24-27, 24-26,24-25, 25-49, 25-48, 25-47, 25-46, 25-45, 25-44, 25-43, 25-42, 25-41,25-40, 25-39, 25-38, 25-37, 25-36, 25-35, 25-34, 25-33, 25-32, 25-31,25-30, 25-29, 25-28, 25-27, 25-26,26-49, 26-48, 26-47, 26-46, 26-45,26-44, 26-43, 26-42, 26-41, 26-40, 26-39, 26-38, 26-37, 26-36, 26-35,26-34, 26-33, 26-32, 26-31, 26-30, 26-29, 26-28, 26-27, 27-49, 27-48,27-47, 27-46, 27-45, 27-44, 27-43, 27-42, 27-41, 27-40, 27-39, 27-38,27-37, 27-36, 27-35, 27-34, 27-33, 27-32, 27-31, 27-30, 27-29, 27-28,28-49, 28-48, 28-47, 28-46, 28-45, 28-44, 28-43, 28-42, 28-41, 28-40,28-39, 28-38, 28-37, 28-36, 28-35, 28-34, 28-33, 28-32, 28-31, 28-30,28-29, 29-49, 29-48, 29-47, 29-46, 29-45, 29-44, 29-43, 29-42, 29-41,29-40, 29-39, 29-38, 29-37, 29-36, 29-35, 29-34, 29-33, 29-32, 29-31,29-30, 30-49, 30-48, 30-47, 30-46, 30-45, 30-44, 30-43, 30-42, 30-41,30-40, 30-39, 30-38, 30-37, 30-36, 30-35, 30-34, 30-33, 30-32, or 30-31nucleotides in length, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50nucleotides in length.

In some embodiments, an antisense polynucleotide agent may comprise acontiguous nucleotide sequence of no more than 22 nucleotides, such asno more than 21 nucleotides, 20 nucleotides, 19 nucleotides, or no morethan 18 nucleotides. In some embodiments the antisense polynucleotideagent of the invention comprises less than 20 nucleotides. In otherembodiments, the antisense polynucleotide agents of the inventioncomprise 20 nucleotides.

In one aspect, an antisense polynucleotide agent of the inventionincludes a sequence selected from the group of sequences provided inTable 3. It will be understood that, although some of the sequences inTable 3 are described as modified and/or conjugated sequences, anantisense polynucleotide agent of the invention, may also comprise anyone of the sequences set forth in Table 3 that is un-modified,un-conjugated, and/or modified and/or conjugated differently thandescribed therein.

By virtue of the nature of the nucleotide sequences provided in Table 3,antisense polynucleotide agents of the invention may include one of thesequences of Table 3 minus only a few nucleotides on one or both endsand yet remain similarly effective as compared to the antisensepolynucleotide agents described above. Hence, antisense polynucleotideagents having a sequence of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14,15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived fromone of the sequences of Table 3 and differing in their ability toinhibit the expression of a LECT2 gene by not more than about 5, 10, 15,20, 25, or 30% inhibition from an antisense polynucleotide agentcomprising the full sequence, are contemplated to be within the scope ofthe present invention.

In addition, the antisense polynucleotide agents provided in Table 3identify a region(s) in a LECT2 transcript that is susceptible toantisense inhibition (e.g., the regions encompassed by the start and endpositions relative to NM_002302.2 in Table 3). As such, the presentinvention further features antisense polynucleotide agents that targetwithin one of these sites. As used herein, an antisense polynucleotideagent is said to target within a particular site of an RNA transcript ifthe antisense polynucleotide agent promotes antisense inhibition of thetarget at that site. Such an antisense polynucleotide agent willgenerally include at least about 15 contiguous nucleotides from one ofthe sequences provided in Table 3 coupled to additional nucleotidesequences taken from the region contiguous to the selected sequence in aLECT2 gene.

While a target sequence is generally about 4-50 nucleotides in length,there is wide variation in the suitability of particular sequences inthis range for directing antisense inhibition of any given target RNA.Various software packages and the guidelines set out herein provideguidance for the identification of optimal target sequences for anygiven gene target, but an empirical approach can also be taken in whicha “window” or “mask” of a given size (as a non-limiting example, 20nucleotides) is literally or figuratively (including, e.g., in silico)placed on the target RNA sequence to identify sequences in the sizerange that can serve as target sequences. By moving the sequence“window” progressively one nucleotide upstream or downstream of aninitial target sequence location, the next potential target sequence canbe identified, until the complete set of possible sequences isidentified for any given target size selected. This process, coupledwith systematic synthesis and testing of the identified sequences (usingassays as described herein or as known in the art) to identify thosesequences that perform optimally can identify those RNA sequences that,when targeted with an antisense polynucleotide agent, mediate the bestinhibition of target gene expression. Thus, while the sequencesidentified, for example, in Table 3 represent effective targetsequences, it is contemplated that further optimization of antisenseinhibition efficiency can be achieved by progressively “walking thewindow” one nucleotide upstream or downstream of the given sequences toidentify sequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified, e.g., inTable 3, further optimization could be achieved by systematically eitheradding or removing nucleotides to generate longer or shorter sequencesand testing those sequences generated by walking a window of the longeror shorter size up or down the target RNA from that point. Again,coupling this approach to generating new candidate targets with testingfor effectiveness of antisense polynucleotide agents based on thosetarget sequences in an inhibition assay as known in the art and/or asdescribed herein can lead to further improvements in the efficiency ofinhibition.

Further still, such optimized sequences can be adjusted by, e.g., theintroduction of modified nucleotides as described herein or as known inthe art, addition or changes in length, or other modifications as knownin the art and/or discussed herein to further optimize the molecule(e.g., increasing serum stability or circulating half-life, increasingthermal stability, enhancing transmembrane delivery, targeting to aparticular location or cell type, increasing interaction with silencingpathway enzymes, increasing release from endosomes) as an expressioninhibitor.

III. Modified Antisense Polynucleotide Agents of the Invention

In one embodiment, the nucleotides of an antisense polynucleotide agentare un-modified, and do not comprise, e.g., chemical modificationsand/or conjugations known in the art and described herein. In anotherembodiment, at least one of the nucleotides of an antisensepolynucleotide agent of the invention is chemically modified to enhancestability or other beneficial characteristics. In certain embodiments ofthe invention, substantially all of the nucleotides of an antisensepolynucleotide agent of the invention are modified. In other embodimentsof the invention, all of the nucleotides of an anti sense polynucleotideagent of the invention are modified. Antisense polynucleotide agents ofthe invention in which “substantially all of the nucleotides aremodified” are largely but not wholly modified and can include not morethan 5, 4, 3, 2, or 1 unmodified nucleotides.

The nucleic acids featured in the invention can be synthesized and/ormodified by standard methods known in the art as further discussedbelow, e.g., solution-phase or solid-phase organic synthesis or both,e.g., by use of an automated DNA synthesizer, such as are commerciallyavailable from, for example, Biosearch, Applied Biosystems, Inc.Well-established methods for the synthesis and/or modification of thenucleic acids featured in the invention are described in, for example,“Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Modifications include, for example,end modifications, e.g., 5′-end modifications (phosphorylation,conjugation, inverted linkages) or 3′-end modifications (conjugation,DNA nucleotides, inverted linkages, etc.); base modifications, e.g.,replacement with stabilizing bases, destabilizing bases, or bases thatbase pair with an expanded repertoire of partners, removal of bases(abasic nucleotides), or conjugated bases; sugar modifications (e.g., atthe 2′-position or 4′-position) or replacement of the sugar; and/orbackbone modifications, including modification or replacement of thephosphodiester linkages.

Specific examples of modified nucleotides useful in the embodimentsdescribed herein include, but are not limited to nucleotides containingmodified backbones or no natural internucleoside linkages. Nucleotideshaving modified backbones include, among others, those that do not havea phosphorus atom in the backbone. For the purposes of thisspecification, and as sometimes referenced in the art, modifiednucleotides that do not have a phosphorus atom in their internucleosidebackbone can also be considered to be oligonucleosides. In someembodiments, a modified antisense polynucleotide agent will have aphosphorus atom in its internucleoside backbone.

Modified nucleotide backbones include, for example, phosphorothioates,chiral phosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6, 239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat.RE39464, the entire contents of each of which are hereby incorporatedherein by reference.

Modified nucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatoms and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, the entire contents of each of which are hereby incorporatedherein by reference.

In other embodiments, suitable nucleotide mimetics are contemplated foruse in antisense polynucleotide agents, in which both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an RNA mimetic that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar backbone of an RNA is replacedwith an amide containing backbone, in particular an aminoethylglycinebackbone. The nucleobases are retained and are bound directly orindirectly to aza nitrogen atoms of the amide portion of the backbone.Representative U.S. patents that teach the preparation of PNA compoundsinclude, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, the entire contents of each of which are herebyincorporated herein by reference. Additional PNA compounds suitable foruse in the antisense polynucleotide agents of the invention aredescribed in, for example, in Nielsen et al., Science, 1991, 254,1497-1500.

Some embodiments featured in the invention include polynucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular——CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂——[known asa methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH2—[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, theantisense polynucleotide agents featured herein have morpholino backbonestructures of the above-referenced U.S. Pat. No. 5,034,506.

Modified nucleotides can also contain one or more modified orsubstituted sugar moieties. The antisense polynucleotide agents featuredherein can include one of the following at the 2′-position: OH; F; O—,S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S— or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Exemplary suitable modifications include O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)nNH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.

In other embodiments, antisense polynucleotide agents include one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br,CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an antisense polynucleotide,or a group for improving the pharmacodynamic properties of an antisensepolynucleotide agent, and other substituents having similar properties.In some embodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)20N(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on a nucleotide of an antisensepolynucleotide agent, particularly the 3′ position of the sugar on the3′ terminal nucleotide. Antisense polynucleotide agents can also havesugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application. The entire contents of eachof the foregoing are hereby incorporated herein by reference.

Additional nucleotides having modified or substituted sugar moieties foruse in the polynucleotide agents of the invention include nucleotidescomprising a bicyclic sugar. A “bicyclic sugar” is a furanosyl ringmodified by the bridging of two atoms. A “bicyclic nucleoside” (“BNA”)is a nucleoside having a sugar moiety comprising a bridge connecting twocarbon atoms of the sugar ring, thereby forming a bicyclic ring system.In certain embodiments, the bridge connects the 4′-carbon and the2′-carbon of the sugar ring. Thus, in some embodiments an antisensepolynucleotide agent may include one or more locked nucleic acids. A“locked nucleic acid” (“LNA”) is a nucleotide having a modified ribosemoiety in which the ribose moiety comprises an extra bridge connectingthe 2′ and 4′ carbons. In other words, an LNA is a nucleotide comprisinga bicyclic sugar moiety comprising a 4′-CH₂—O-2′ bridge. This structureeffectively “locks” the ribose in the 3′-endo structural conformation.The addition of locked nucleic acids to santisense polynucleotide agentshas been shown to increase santisense polynucleotide agent stability inserum, and to reduce off-target effects (Elmen, J. et al., (2005)Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol CancTher 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research31(12):3185-3193).

Examples of bicyclic nucleosides for use in the polynucleotides of theinvention include without limitation nucleosides comprising a bridgebetween the 4′ and the 2′ ribosyl ring atoms. In certain embodiments,the antisense polynucleotide agents of the invention include one or morebicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′to 2′ bridged bicyclic nucleosides, include but are not limited to4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′; 4′-(CH₂)2-O-2′ (ENA);4′-CH(CH₃)—O-2′ (also referred to as “constrained ethyl” or “cEt”) and4′-CH(CH₂OCH₃)-O-2′ (and analogs thereof; see, e.g., U.S. Pat. No.7,399,845); 4′-C(CH₃)(CH₃)—O-2′ (and analogs thereof; see e.g., U.S.Pat. No. 8,278,283); 4′-CH₂—N(OCH₃)-2′ (and analogs thereof; see e.g.,U.S. Pat. No. 8,278,425); 4′-CH₂—O—N(CH₃)-2′ (see, e.g., U.S. PatentPublication No. 2004/0171570); 4′-CH₂—N(R)—O-2′, wherein R is H, C1-C12alkyl, or a protecting group (see, e.g., U.S. Pat. No. 7,427,672);4′-CH₂—C(H)(CH₃)-2′ (see, e.g., Chattopadhyaya et al., J. Org. Chem.,2009, 74, 118-134); and 4′-CH₂—C(═CH2)-2′ (and analogs thereof; see,e.g., U.S. Pat. No. 8,278,426). The entire contents of each of theforegoing are hereby incorporated herein by reference.

Additional representative U.S. Patents and US Patent Publications thatteach the preparation of locked nucleic acid nucleotides include, butare not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191;6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207;7,034,133;7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457;8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618;and US 2009/0012281, the entire contents of each of which are herebyincorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one ormore stereochemical sugar configurations including for exampleα-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

In one particular embodiment of the invention, an antisensepolynucleotide agent can include one or more constrained ethylnucleotides. As used herein, a “constrained ethyl nucleotide” or “cEt”is a locked nucleic acid comprising a bicyclic sugar moiety comprising a4′-CH(CH₃)—O-2′ bridge. In one embodiment, a constrained ethylnucleotide is in an S conformation and is referred to as an“S-constrained ethyl nucleotide” or “S-cEt.”

Modified nucleotides included in the antisense polynucleotide agents ofthe invention can also contain one or more sugar mimetics. For example,the antisense polynucleotide agent may include a “modifiedtetrahydropyran nucleotide” or “modified THP nucleotide.” A “modifiedtetrahydropyran nucleotide” has a six-membered tetrahydropyran “sugar”substituted in for the pentofuranosyl residue in normal nucleotides (asugar surrogate). Modified THP nucleotides include, but are not limitedto, what is referred to in the art as hexitol nucleic acid (HNA), anitolnucleic acid (ANA), manitol nucleic acid (MNA) (see, e.g., Leumann,Bioorg. Med. Chem., 2002, 10, 841-854), or fluoro HNA (F-HNA).

In some embodiments of the invention, sugar surrogates comprise ringshaving more than 5 atoms and more than one heteroatom. For examplenucleotides comprising morpholino sugar moieties and their use inoligomeric compounds has been reported (see for example: Braasch et al.,Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos. 5,698,685;5,166,315; 5,185,444; and 5,034,506). Morpholinos may be modified, forexample by adding or altering various substituent groups from the abovemorpholino structure. Such sugar surrogates are referred to herein as“modified morpholinos.”

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see PCT InternationalApplication WO 2008/101157 published on Aug. 21, 2008 for otherdisclosed 5′, 2′-bis substituted nucleosides) and replacement of theribosyl ring oxygen atom with S and further substitution at the2′-position (see published U.S. Patent Application US2005-0130923,published on Jun. 16, 2005) or alternatively 5′-substitution of abicyclic nucleic acid (see PCT International Application WO 2007/134181,published on 11/22/07 wherein a 4′-CH₂-O-2′ bicyclic nucleoside isfurther substituted at the 5′ position with a 5′-methyl or a 5′-vinylgroup). The synthesis and preparation of carbocyclic bicyclicnucleosides along with their oligomerization and biochemical studieshave also been described (see, e.g., Srivastava et al., J. Am. Chem.Soc. 2007, 129(26), 8362-8379).

In certain embodiments, antisense compounds comprise one or moremodified cyclohexenyl nucleosides, which is a nucleoside having asix-membered cyclohexenyl in place of the pentofuranosyl residue innaturally occurring nucleosides. Modified cyclohexenyl nucleosidesinclude, but are not limited to those described in the art (see forexample commonly owned, published PCT Application WO 2010/036696,published on Apr. 10, 2010, Robeyns et al., J. Am. Chem. Soc., 2008,130(6), 1979-1984; Horvath et al., Tetrahedron Letters, 2007, 48,3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30),9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005,24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005,33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F:Structural Biology and Crystallization Communications, 2005, F61(6),585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al.,Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem.,2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001,29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wanget al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7),785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published PCTapplication, WO 06/047842; and Published PCT Application WO 01/049687;the text of each is incorporated by reference herein, in theirentirety).

An antisense polynucleotide agent can also include nucleobasemodifications or substitutions. As used herein, “unmodified” or“natural” nucleobases include the purine bases adenine (A) and guanine(G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).Modified nucleobases include other synthetic and natural nucleobasessuch as deoxy-thymine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl analother 8-substituted adenines and guanines, 5-halo, particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine.

Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808,those disclosed in “Modified Nucleosides in Biochemistry,” Biotechnologyand Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in TheConcise Encyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y S., Chapter 15, antisensepolynucleotide agent Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theagents featured in the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., Eds., antisense polynucleotide agent Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and areexemplary base substitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941;5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887;6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and7,495,088, the entire contents of each of which are hereby incorporatedherein by reference.

One or more of the nucleotides of an iRNA of the invention may alsoinclude a hydroxymethyl substituted nucleotide. A “hydroxymethylsubstituted nucleotide” is an acyclic 2′-3′-seco-nucleotide, alsoreferred to as an “unlocked nucleic acid” (“UNA”) modification.Representative U.S. publications that teach the preparation of UNAinclude, but are not limited to, U.S. Pat. No. 8,314,227; and US PatentPublication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, theentire contents of each of which are hereby incorporated herein byreference.

Additional modification which may potentially stabilize the ends ofantisense polynucleotide agents can includeN-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc),N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol(Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether),N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others.Disclosure of this modification can be found in US Patent PublicationNo. 2012/0142101.

Any of the antisense polynucleotide agents of the invention may beoptionally conjugated with a GalNAc derivative ligand, as described inSection IV, below.

As described in more detail below, an agent that contains conjugationsof one or more carbohydrate moieties to an antisense polynucleotideagent can optimize one or more properties of the agent. In many cases,the carbohydrate moiety will be attached to a modified subunit of theantisense polynucleotide agent. For example, the ribose sugar of one ormore ribonucleotide subunits of an agent can be replaced with anothermoiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which isattached a carbohydrate ligand. A ribonucleotide subunit in which theribose sugar of the subunit has been so replaced is referred to hereinas a ribose replacement modification subunit (RRMS). A cyclic carriermay be a carbocyclic ring system, i.e., all ring atoms are carbon atoms,or a heterocyclic ring system, i.e., one or more ring atoms may be aheteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be amonocyclic ring system, or may contain two or more rings, e.g. fusedrings. The cyclic carrier may be a fully saturated ring system, or itmay contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,”preferably two “backbone attachment points” and (ii) at least one“tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide and polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

The antisense polynucleotide agents may be conjugated to a ligand via acarrier, wherein the carrier can be cyclic group or acyclic group;preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl,pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl,[1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl,thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl,tetrahydrofuryl and and decalin; preferably, the acyclic group isselected from serinol backbone or diethanolamine backbone.

In certain specific embodiments, the antisense polynucleotide agent foruse in the methods of the invention is an agent selected from the groupof agents listed in Table 3. These agents may further comprise a ligand,as described in Section IV, below.

A. Antisense Polynucleotide Agents Comprising Motifs

In certain embodiments of the invention, at least one of the contiguousnucleotides of the antisense polynucleotide agents of the invention maybe a modified nucleotide. In one embodiment, the modified nucleotidecomprises one or more modified sugars. In other embodiments, themodified nucleotide comprises one or more modified nucleobases. In yetother embodiments, the modified nucleotide comprises one or moremodified internucleoside linkages. In some embodiments, themodifications (sugar modifications, nucleobase modifications, and/orlinkage modifications) define a pattern or motif. In one embodiment, thepatterns of modifications of sugar moieties, internucleoside linkages,and nucleobases are each independent of one another.

Antisense polynucleotide agents having modified oligonucleotidesarranged in patterns, or motifs may, for example, confer to the agentsproperties such as enhanced inhibitory activity, increased bindingaffinity for a target nucleic acid, or resistance to degradation by invivo nucleases. For example, such agents may contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, increased binding affinity for the targetnucleic acid, and/or increased inhibitory activity. A second region ofsuch agents may optionally serve as a substrate for the cellularendonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

An exemplary antisense polynucleotide agent having modifiedoligonucleotides arranged in patterns, or motifs is a gapmer. In a“gapmer”, an internal region or “gap” having a plurality of linkednucleotides that supports RNaseH cleavage is positioned between twoexternal flanking regions or “wings” having a plurality of linkednucleotides that are chemically distinct from the linked nucleotides ofthe internal region. The gap segment generally serves as the substratefor endonuclease cleavage, while the wing segments comprise modifiednucleotides.

The three regions of a gapmer motif (the 5 ′-wing, the gap, and the 3′-wing) form a contiguous sequence of nucleotides and may be describedas “X-Y-Z”, wherein “X” represents the length of the 5-wing, “Y”represents the length of the gap, and “Z” represents the length of the3′-wing. In one embodiment, a gapmer described as “X-Y-Z” has aconfiguration such that the gap segment is positioned immediatelyadjacent to each of the 5′ wing segment and the 3′ wing segment. Thus,no intervening nucleotides exist between the 5′ wing segment and gapsegment, or the gap segment and the 3′ wing segment. Any of theantisense compounds described herein can have a gapmer motif. In someembodiments, X and Z are the same, in other embodiments they aredifferent.

In certain embodiments, the regions of a gapmer are differentiated bythe types of modified nucleotides in the region. The types of modifiednucleotides that may be used to differentiate the regions of a gapmer,in some embodiments, include 0-D-ribonucleotides,0-D-deoxyribonucleotides, 2′-modified nucleotides, e.g., 2′-modifiednucleotides (e.g., 2′-MOE, and 2′-O—CH₃), and bicyclic sugar modifiednucleotides (e.g., those having a 4′-(CH₂)n-O-2′ bridge, where n=1 orn=2).

In one embodiment, at least some of the modified nucleotides of each ofthe wings may differ from at least some of the modified nucleotides ofthe gap. For example, at least some of the modified nucleotides of eachwing that are closest to the gap (the 3 ‘-most nucleotide of the 5’-wingand the 5′-most nucleotide of the 3 -wing) differ from the modifiednucleotides of the neighboring gap nucleotides, thus defining theboundary between the wings and the gap. In certain embodiments, themodified nucleotides within the gap are the same as one another. Incertain embodiments, the gap includes one or more modified nucleotidesthat differ from the modified nucleotides of one or more othernucleotides of the gap.

The length of the 5′-wing (X) of a gapmer may be 1 to 6 nucleotides inlength, e.g., 2 to 6, 2 to 5, 3 to 6, 3 to 5, 1 to 5, 1 to 4, 1 to 3, 2to 4 nucleotides in length, e.g., 1, 2, 3, 4, 5, or 6 nucleotides inlength.

The length of the 3′-wing (Z) of a gapmer may be 1 to 6 nucleotides inlength, e.g., 2 to 6, 2-5, 3 to 6, 3 to 5, 1 to 5, 1 to 4, 1 to 3, 2 to4 nucleotides in length, e.g., 1, 2, 3, 4, 5, or 6 nucleotides inlength.

The length of the gap (Y) of a gapmer may be 5 to 14 nucleotides inlength, e.g., 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 5 to 8, 5 to7, 5 to 6, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 6 to 9, 6 to 8,6 to 7, 7 to 14, 7 to 13, 7 to 12, 7 to 11, 7 to 10, 7 to 9, 7 to 8, 8to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 8 to 9, 9 to 14, 9 to 13, 9to 12, 9 to 11, 9 to 10, 10 to 14, 10 to 13, 10 to 12, 10 to 11, 11 to14, 11 to 13, 11 to 12, 12 to 14, 12 to 13, or 13 to 14 nucleotides inlength, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides inlength.

In some embodiments of the invention X consists of 2, 3, 4, 5 or 6nucleotides, Y consists of 7, 8, 9, 10, 11, or 12 nucleotides, and Zconsists of 2, 3, 4, 5 or 6 nucleotides. Such gapmers include (X-Y-Z)2-7-2, 2-7-3, 2-7-4, 2-7-5, 2-7-6, 3-7-2, 3-7-3, 3-7-4, 3-7-5, 3-7-6,4-7-3, 4-7-4, 4-7-5, 4-7-6, 5-7-3, 5-7-4, 5-7-5, 5-7-6, 6-7-3, 6-7-4,6-7-5, 6-7-6, 3-7-3, 3-7-4, 3-7-5, 3-7-6, 4-7-3, 4-7-4, 4-7-5, 4-7-6,5-7-3, 5-7-4, 5-7-5, 5-7-6, 6-7-3, 6-7-4, 6-7-5, 6-7-6, 2-8-2, 2-8-3,2-8-4, 2-8-5, 2-8-6, 3-8-2, 3-8-3, 3-8-4, 3-8-5, 3-8-6, 4-8-3, 4-8-4,4-8-5, 4-8-6, 5-8-3, 5-8-4, 5-8-5, 5-8-6, 6-8-3, 6-8-4, 6-8-5, 6-8-6,2-9-2, 2-9-3, 2-9-4, 2-9-5, 2-9-6, 3-9-2, 3-9-3, 3-9-4, 3-9-5, 3-9-6,4-9-3, 4-9-4, 4-9-5, 4-9-6, 5-9-3, 5-9-4, 5-9-5, 5-9-6, 6-9-3, 6-9-4,6-9-5, 6-9-6, 2-10-2, 2-10-3, 2-10-4, 2-10-5, 2-10-6, 3-10-2, 3-10-3,3-10-4, 3-10-5, 3-10-6, 4-10-3, 4-10-4, 4-10-5, 4-10-6, 5-10-3, 5-10-4,5-10-5, 5-10-6, 6-10-3, 6-10-4, 6-10-5, 6-10-6, 2-11-2, 2-11-3, 2-11-4,2-11-5, 2-11-6, 3-11-2, 3-11-3, 3-11-4, 3-11-5, 3-11-6, 4-11-3, 4-11-4,4-11-5, 4-11-6, 5-11-3, 5-11-4, 5-11-5, 5-11-6, 6-11-3, 6-11-4, 6-11-5,6-11-6, 2-12-2, 2-12-3, 2-12-4, 2-12-5, 2-12-6, 3-12-2, 3-12-3, 3-12-4,3-12-5, 3-12-6, 4-12-3, 4-12-4, 4-12-5, 4-12-6, 5-12-3, 5-12-4, 5-12-5,5-12-6, 6-12-3, 6-12-4, 6-12-5, or 6-12-6.

In some embodiments of the invention, antisense polynucleotide agentstargeting LECT2 include a 5-10-5 gapmer motif. In other embodiments ofthe invention, antisense polynucleotide agents targeting LECT2 include a4-10-4 gapmer motif In another embodiment of the invention, antisensepolynucleotide agents targeting LECT2 include a 3-10-3 gapmer motif. Inyet other embodiments of the invention, antisense polynucleotide agentstargeting LECT2 include a 2-10-2 gapmer motif.

The 5′-wing and/or 3′-wing of a gapmer may independently include 1-6modified nucleotides, e.g., 1, 2, 3, 4, 5, or 6 modified nucleotides.

In some embodiment, the 5′-wing of a gapmer includes at least onemodified nucleotide. In one embodiment, the 5′-wing of a gapmercomprises at least two modified nucleotides. In another embodiment, the5′-wing of a gapmer comprises at least three modified nucleotides. Inyet another embodiment, the 5′-wing of a gapmer comprises at least fourmodified nucleotides. In another embodiment, the 5′-wing of a gapmercomprises at least five modified nucleotides. In certain embodiments,each nucleotide of the 5′-wing of a gapmer is a modified nucleotide.

In some embodiments, the 3′-wing of a gapmer includes at least onemodified nucleotide. In one embodiment, the 3′-wing of a gapmercomprises at least two modified nucleotides. In another embodiment, the3′-wing of a gapmer comprises at least three modified nucleotides. Inyet another embodiment, the 3′-wing of a gapmer comprises at least fourmodified nucleotides. In another embodiment, the 3′-wing of a gapmercomprises at least five modified nucleotides. In certain embodiments,each nucleotide of the 3′-wing of a gapmer is a modified nucleotide.

In certain embodiments, the regions of a gapmer are differentiated bythe types of sugar moieties of the nucleotides. In one embodiment, thenucleotides of each distinct region comprise uniform sugar moieties. Inother embodiments, the nucleotides of each distinct region comprisedifferent sugar moieties. In certain embodiments, the sugar nucleotidemodification motifs of the two wings are the same as one another. Incertain embodiments, the sugar nucleotide modification motifs of the5′-wing differs from the sugar nucleotide modification motif of the3′-wing.

The 5′-wing of a gapmer may include 1-6 modified nucleotides, e.g., 1,2, 3, 4, 5, or 6 modified nucleotides.

In one embodiment, at least one modified nucleotide of the 5′-wing of agapmer is a bicyclic nucleotide, such as a constrained ethyl nucleotide,or an LNA. In another embodiment, the 5′-wing of a gapmer includes 2, 3,4, or 5 bicyclic nucleotides. In some embodiments, each nucleotide ofthe 5′-wing of a gapmer is a bicyclic nucleotide.

In one embodiment, the 5′-wing of a gapmer includes at least 1, 2, 3, 4,or 5 constrained ethyl nucleotides. In some embodiments, each nucleotideof the 5′-wing of a gapmer is a constrained ethyl nucleotide.

In one embodiment, the 5′-wing of a gapmer comprises at least one LNAnucleotide. In another embodiment, the 5′-wing of a gapmer includes 2,3, 4, or 5 LNA nucleotides. In other embodiments, each nucleotide of the5′-wing of a gapmer is an LNA nucleotide.

In certain embodiments, at least one modified nucleotide of the 5′-wingof a gapmer is a non-bicyclic modified nucleotide, e.g., a2′-substituted nucleotide. A “2′-substituted nucleotide” is a nucleotidecomprising a modification at the 2′-position which is other than H orOH, such as a 2′-OMe nucleotide, or a 2′-MOE nucleotide. In oneembodiment, the 5′-wing of a gapmer comprises 2, 3, 4, or 52′-substituted nucleotides. In one embodiment, each nucleotide of the5′-wing of a gapmer is a 2′-substituted nucleotide.

In one embodiment, the 5′-wing of a gapmer comprises at least one 2′-OMenucleotide. In one embodiment, the 5′-wing of a gapmer comprises atleast 2, 3, 4, or 5 2′-OMe nucleotides. In one embodiment, each of thenucleotides of the 5′-wing of a gapmer comprises a 2′-OMe nucleotide.

In one embodiment, the 5′-wing of a gapmer comprises at least one 2′-MOEnucleotide. In one embodiment, the 5′-wing of a gapmer comprises atleast 2, 3, 4, or 5 2′-MOE nucleotides. In one embodiment, each of thenucleotides of the 5′-wing of a gapmer comprises a 2′-MOE nucleotide.

In certain embodiments, the 5′-wing of a gapmer comprises at least one2′-deoxynucleotide. In certain embodiments, each nucleotide of the5′-wing of a gapmer is a 2′-deoxynucleotide. In a certain embodiments,the 5′-wing of a gapmer comprises at least one ribonucleotide. Incertain embodiments, each nucleotide of the 5′-wing of a gapmer is aribonucleotide.

The 3′-wing of a gapmer may include 1-6 modified nucleotides, e.g., 1,2, 3, 4, 5, or 6 modified nucleotides.

In one embodiment, at least one modified nucleotide of the 3′-wing of agapmer is a bicyclic nucleotide, such as a constrained ethyl nucleotide,or an LNA. In another embodiment, the 3′-wing of a gapmer includes 2, 3,4, or 5 bicyclic nucleotides. In some embodiments, each nucleotide ofthe 3′-wing of a gapmer is a bicyclic nucleotide.

In one embodiment, the 3′-wing of a gapmer includes at least oneconstrained ethyl nucleotide. In another embodiment, the 3′-wing of agapmer includes 2, 3, 4, or 5 constrained ethyl nucleotides. In someembodiments, each nucleotide of the 3′-wing of a gapmer is a constrainedethyl nucleotide.

In one embodiment, the 3′-wing of a gapmer comprises at least one LNAnucleotide. In another embodiment, the 3′-wing of a gapmer includes 2,3, 4, or 5 LNA nucleotides. In other embodiments, each nucleotide of the3′-wing of a gapmer is an LNA nucleotide.

In certain embodiments, at least one modified nucleotide of the 3′-wingof a gapmer is a non-bicyclic modified nucleotide, e.g., a2′-substituted nucleotide. In one embodiment, the 3′-wing of a gapmercomprises 2, 3, 4, or 5 2′-substituted nucleotides. In one embodiment,each nucleotide of the 3′-wing of a gapmer is a 2′-substitutednucleotide.

In one embodiment, the 3′-wing of a gapmer comprises at least one 2′-0Menucleotide. In one embodiment, the 3′-wing of a gapmer comprises atleast 2, 3, 4, or 5 2′-OMe nucleotides. In one embodiment, each of thenucleotides of the 3′-wing of a gapmer comprises a 2′-OMe nucleotide.

In one embodiment, the 3′-wing of a gapmer comprises at least one 2′-MOEnucleotide. In one embodiment, the 3′-wing of a gapmer comprises atleast 2, 3, 4, or 5 2′-MOE nucleotides. In one embodiment, each of thenucleotides of the 3′-wing of a gapmer comprises a 2′-MOE nucleotide.

In certain embodiments, the 3′-wing of a gapmer comprises at least one2′-deoxynucleotide. In certain embodiments, each nucleotide of the3′-wing of a gapmer is a 2′-deoxynucleotide. In a certain embodiments,the 3′-wing of a gapmer comprises at least one ribonucleotide. Incertain embodiments, each nucleotide of the 3′-wing of a gapmer is aribonucleotide.

The gap of a gapmer may include 5-14 modified nucleotides, e.g., 5, 6,7, 8, 9, 10, 11, 12, 13, or 14 modified nucleotides.

In one embodiment, the gap of a gapmer comprises at least one5-methylcytosine. In one embodiment, the gap of a gapmer comprises atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 5-methylcytosines. Inone embodiment, all of the nucleotides of the the gap of a gapmer are5-methylcytosines.

In one embodiment, the gap of a gapmer comprises at least one2′-deoxynucleotide. In one embodiment, the gap of a gapmer comprises atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 2′-deoxynucleotides. Inone embodiment, all of the nucleotides of the the gap of a gapmer are2′-deoxynucleotides.

A gapmer may include one or more modified internucleotide linkages. Insome embodiments, a gapmer includes one or more phosphodiesterinternucleotide linkages. In other embodiments, a gapmer includes one ormore phosphorothioate internucleotide linkages.

In one embodiment, each nucleotide of a 5′-wing of a gapmer are linkedvia a phosphorothioate internucleotide linkage. In another embodiment,each nucleotide of a 3′-wing of a gapmer are linked via aphosphorothioate internucleotide linkage. In yet another embodiment,each nucleotide of a gap segment of a gapmer is linked via aphosphorothioate internucleotide linkage. In one embodiment, all of thenucleotides in a gapmer are linked via phosphorothioate internucleotidelinkages.

In one embodiment, an antisense polynucleotide agent targeting a LECT2gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fivenucleotides and a 3′-wing segment comprising 5 nucleotides.

In another embodiment, an antisense polynucleotide agent targeting aLECT2 gene comprises a gap segment of ten 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising four nucleotides and a 3′-wing segment comprising fournucleotides.

In another embodiment, an antisense polynucleotide agent targeting aLECT2 gene comprises a gap segment of ten 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising three nucleotides and a 3′-wing segment comprising threenucleotides.

In another embodiment, an antisense polynucleotide agent targeting aLECT2 gene comprises a gap segment of ten 2′-deoxyribonucleotidespositioned immediately adjacent to and between a 5′-wing segmentcomprising two nucleotides and a 3′-wing segment comprising twonucleotides.

In one embodiment, each nucleotide of a 5-wing flanking a gap segment of10 2′-deoxyribonucleotides comprises a modified nucleotide. In anotherembodiment, each nucleotide of a 3-wing flanking a gap segment of 102′-deoxyribonucleotides comprises a modified nucleotide. In oneembodiment, each of the modified 5′-wing nucleotides and each of themodified 3′-wing nucleotides comprise a 2′-sugar modification. In oneembodiment, the 2′-sugar modification is a 2′-OMe modification. Inanother embodiment, the 2′-sugar modification is a 2′-MOE modification.In one embodiment, each of the modified 5′-wing nucleotides and each ofthe modified 3′-wing nucleotides comprise a bicyclic nucleotide. In oneembodiment, the bicyclic nucleotide is a constrained ethyl nucleotide.In another embodiment, the bicyclic nucleotide is an LNA nucleotide. Inone embodiment, each cytosine in an antisense polynucleotide agenttargeting a LECT2 gene is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting a LECT2gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fivenucleotides comprising a 2′OMe modification and a 3′-wing segmentcomprising five nucleotides comprising a 2′OMe modification, whereineach internucleotde linkage of the agent is a phosphorothioate linkage.In one embodiment, each cytosine of the agent is a 5-methylcytosine. Inone embodiment, the agent further comprises a ligand.

In one embodiment, an antisense polynucleotide agent targeting a LECT2gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fivenucleotides comprising a 2′MOE modification and a 3′-wing segmentcomprising five nucleotides comprising a 2′MOE modification, whereineach internucleotide linkage of the agent is a phosphorothioate linkage.In one embodiment, each cytosine of the agent is a 5-methylcytosine. Inone embodiment, the agent further comprises a ligand.

In one embodiment, an antisense polynucleotide agent targeting a LECT2gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fiveconstrained ethyl nucleotides and a 3′-wing segment comprising fiveconstrained ethyl nucleotides, wherein each internucleoitde linkage ofthe agent is a phosphorothioate linkage. In one embodiment, eachcytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting a LECT2gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fiveLNA nucleotides and a 3′-wing segment comprising five LNA nucleotides,wherein each internucleotide linkage of the agent is a phosphorothioatelinkage. In one embodiment, each cytosine of the agent is a5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting a LECT2gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fournucleotides comprising a 2′OMe modification and a 3′-wing segmentcomprising four nucleotides comprising a 2′OMe modification, whereineach internucleotde linkage of the agent is a phosphorothioate linkage.In one embodiment, each cytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting a LECT2gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fournucleotides comprising a 2′MOE modification and a 3′-wing segmentcomprising four nucleotides comprising a 2′MOE modification, whereineach internucleotide linkage of the agent is a phosphorothioate linkage.In one embodiment, each cytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting a LECT2gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fourconstrained ethyl nucleotides and a 3′-wing segment comprising fourconstrained ethyl nucleotides, wherein each internucleoitde linkage ofthe agent is a phosphorothioate linkage. In one embodiment, eachcytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting a LECT2gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising fourLNA nucleotides and a 3′-wing segment comprising four LNA nucleotides,wherein each internucleotide linkage of the agent is a phosphorothioatelinkage. In one embodiment, each cytosine of the agent is a5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting a LECT2gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising threenucleotides comprising a 2′OMe modification and a 3′-wing segmentcomprising three nucleotides comprising a 2′OMe modification, whereineach internucleotde linkage of the agent is a phosphorothioate linkage.In one embodiment, each cytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting a LECT2gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising threenucleotides comprising a 2′MOE modification and a 3′-wing segmentcomprising three nucleotides comprising a 2′MOE modification, whereineach internucleotide linkage of the agent is a phosphorothioate linkage.In one embodiment, each cytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting a LECT2gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising threeconstrained ethyl nucleotides and a 3′-wing segment comprising threeconstrained ethyl nucleotides, wherein each internucleoitde linkage ofthe agent is a phosphorothioate linkage. In one embodiment, eachcytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting a LECT2gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising threeLNA nucleotides and a 3′-wing segment comprising three LNA nucleotides,wherein each internucleotide linkage of the agent is a phosphorothioatelinkage. In one embodiment, each cytosine of the agent is a5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting a LECT2gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising twonucleotides comprising a 2′OMe modification and a 3′-wing segmentcomprising two nucleotides comprising a 2′OMe modification, wherein eachinternucleotde linkage of the agent is a phosphorothioate linkage. Inone embodiment, each cytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting a LECT2gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising twonucleotides comprising a 2′MOE modification and a 3′-wing segmentcomprising two nucleotides comprising a 2′MOE modification, wherein eachinternucleotide linkage of the agent is a phosphorothioate linkage. Inone embodiment, each cytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting a LECT2gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising twoconstrained ethyl nucleotides and a 3′-wing segment comprising twoconstrained ethyl nucleotides, wherein each internucleoitde linkage ofthe agent is a phosphorothioate linkage. In one embodiment, eachcytosine of the agent is a 5-methylcytosine.

In one embodiment, an antisense polynucleotide agent targeting a LECT2gene comprises a gap segment of ten 2′-deoxyribonucleotides positionedimmediately adjacent to and between a 5′-wing segment comprising two LNAnucleotides and a 3′-wing segment comprising two LNA nucleotides,wherein each internucleotide linkage of the agent is a phosphorothioatelinkage. In one embodiment, each cytosine of the agent is a5-methylcytosine.

Further gapmer designs suitable for use in the agents, compositions, andmethods of the invention are disclosed in, for example, U.S. Pat. Nos.7,687,617 and 8,580,756; U.S. Patent Publication Nos. 20060128646,20090209748, 20140128586, 20140128591, 20100210712, and 20080015162A1;and International Publication No. WO 2013/159108, the entire content ofeach of which are incorporated herein by reference.

IV. Antisense Polynucleotide Agents Conjugated to Ligands

Another modification of the polynucleotide agents of the inventioninvolves chemically linking to the agent one or more ligands, moietiesor conjugates that enhance the activity, cellular distribution orcellular uptake of the antisense polynucleotide agent. Such moietiesinclude but are not limited to lipid moieties such as a cholesterolmoiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994,4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al.,Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med.Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991,10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuket al., Biochimie, 1993, 75:49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res.,1990, 18:3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In one embodiment, a ligand alters the distribution, targeting orlifetime of an antisense polynucleotide agent into which it isincorporated. In preferred embodiments a ligand provides an enhancedaffinity for a selected target, e.g., molecule, cell or cell type,compartment, e.g., a cellular or organ compartment, tissue, organ orregion of the body, as, e.g., compared to a species absent such aligand. Preferred ligands will not take part in hybridization of anantisense polynucleotide agent to the targeted mRNA.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or alipid. The ligand can also be a recombinant or synthetic molecule, suchas a synthetic polymer, e.g., a synthetic polyamino acid. Examples ofpolyamino acids include polyamino acid is a polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucoseamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGDpeptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a hepaticcell. Ligands can also include hormones and hormone receptors. They canalso include non-peptidic species, such as lipids, lectins,carbohydrates, vitamins, cofactors, multivalent lactose, multivalentgalactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalentmannose, or multivalent fucose. The ligand can be, for example, alipopolysaccharide, an activator of p38 MAP kinase, or an activator ofNF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the antisense polynucleotide agent into the cell, for example,by disrupting the cell's cytoskeleton, e.g., by disrupting the cell'smicrotubules, microfilaments, and/or intermediate filaments. The drugcan be, for example, taxon, vincristine, vinblastine, cytochalasin,nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A,indanocine, or myoservin.

In some embodiments, a ligand attached to an antisense polynucleotideagent as described herein acts as a pharmacokinetic modulator (PKmodulator). PK modulators include lipophiles, bile acids, steroids,phospholipid analogues, peptides, protein binding agents, PEG, vitaminsetc. Exemplary PK modulators include, but are not limited to,cholesterol, fatty acids, cholic acid, lithocholic acid,dialkylglycerides, diacylglyceride, phospholipids, sphingolipids,naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides thatcomprise a number of phosphorothioate linkages are also known to bind toserum protein, thus short oligonucleotides, e.g., oligonucleotides ofabout 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple ofphosphorothioate linkages in the backbone are also amenable to thepresent invention as ligands (e.g. as PK modulating ligands). Inaddition, aptamers that bind serum components (e.g. serum proteins) arealso suitable for use as PK modulating ligands in the embodimentsdescribed herein.

Ligand-conjugated polynucleotides of the invention may be synthesized bythe use of a polynucleotide that bears a pendant reactive functionality,such as that derived from the attachment of a linking molecule onto theoligonucleotide (described below). This reactive polynucleotide may bereacted directly with commercially-available ligands, ligands that aresynthesized bearing any of a variety of protecting groups, or ligandsthat have a linking moiety attached thereto.

The polynucleotides used in the conjugates of the present invention maybe conveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other polynucleotides, such as thephosphorothioates and alkylated derivatives.

In the ligand-conjugated polynucleotides and ligand-molecule bearingsequence-specific linked nucleosides of the present invention, thepolynucleotides and polynucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide. In someembodiments, the polynucleotides or linked nucleosides of the presentinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

A. Lipid Conjugates

In one embodiment, the ligand or conjugate is a lipid or lipid-basedmolecule. Such a lipid or lipid-based molecule preferably binds a serumprotein, e.g., human serum albumin (HSA). An HSA binding ligand allowsfor distribution of the conjugate to a target tissue, e.g., a non-kidneytarget tissue of the body. For example, the target tissue can be theliver, including parenchymal cells of the liver. Other molecules thatcan bind HSA can also be used as ligands. For example, naproxen oraspirin can be used. A lipid or lipid-based ligand can (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) can be used toadjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bytarget cells such as liver cells. Also included are HSA and low densitylipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to antisensepolynucleotide agents can affect pharmacokinetic distribution of theagent, such as by enhancing cellular recognition and absorption. Thepeptide or peptidomimetic moiety can be about 5-50 amino acids long,e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO: 9). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO: 10) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12)have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to anantisens epolynucleotide agent via an incorporated monomer unit for celltargeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide,or RGD mimic. A peptide moiety can range in length from about 5 aminoacids to about 40 amino acids. The peptide moieties can have astructural modification, such as to increase stability or directconformational properties. Any of the structural modifications describedbelow can be utilized.

An RGD peptide for use in the compositions and methods of the inventionmay be linear or cyclic, and may be modified, e.g., glycosylated ormethylated, to facilitate targeting to a specific tissue(s).RGD-containing peptides and peptidiomimemtics may include D-amino acids,as well as synthetic RGD mimics. In addition to RGD, one can use othermoieties that target the integrin ligand. Preferred conjugates of thisligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an a-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., a -defensin, (3-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, anantisense polynucleotide agent further comprises a carbohydrate. Thecarbohydrate conjugated agents are advantageous for the in vivo deliveryof nucleic acids, as well as compositions suitable for in vivotherapeutic use, as described herein (see, e.g., Prakash, et al. (2014)Nuc Acid Res doi 10.1093/nar/gku531). As used herein, “carbohydrate”refers to a compound which is either a carbohydrate per se made up ofone or more monosaccharide units having at least 6 carbon atoms (whichcan be linear, branched or cyclic) with an oxygen, nitrogen or sulfuratom bonded to each carbon atom; or a compound having as a part thereofa carbohydrate moiety made up of one or more monosaccharide units eachhaving at least six carbon atoms (which can be linear, branched orcyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbonatom. Representative carbohydrates include the sugars (mono-, di-, tri-and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9monosaccharide units), and polysaccharides such as starches, glycogen,cellulose and polysaccharide gums. Specific monosaccharides include C5and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharidesinclude sugars having two or three monosaccharide units (e.g., C5, C6,C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositionsand methods of the invention is a monosaccharide. In some embodiments,the GalNAc conjugate is

In some embodiments, a carbohydrate conjugate for use in thecompositions and methods of the invention is selected from the groupconsisting of:

Another representative carbohydrate conjugate for use in the embodimentsdescribed herein includes, but is not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one ormore additional ligands as described above, such as, but not limited to,a PK modulator and/or a cell permeation peptide.

D. Linkers

In some embodiments, the conjugate or ligand described herein can beattached to an antisense polynucleotide agent with various linkers thatcan be cleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound, e.g., covalently attaches two parts ofa compound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic orsubstituted aliphatic. In one embodiment, the linker is between about1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17,8-17, 6-16, 7-16, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least about 10 times, 20,times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times or more, or at least about 100 times faster in a target cell orunder a first reference condition (which can, e.g., be selected to mimicor represent intracellular conditions) than in the blood of a subject,or under a second reference condition (which can, e.g., be selected tomimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing a cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, aliver-targeting ligand can be linked to a cationic lipid through alinker that includes an ester group. Liver cells are rich in esterases,and therefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus, one can determine the relative susceptibilityto cleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It can be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, orabout 100 times faster in the cell (or under in vitro conditionsselected to mimic intracellular conditions) as compared to blood orserum (or under in vitro conditions selected to mimic extracellularconditions).

i. Redox Cleavable Linking Groups

In one embodiment, a cleavable linking group is a redox cleavablelinking group that is cleaved upon reduction or oxidation. An example ofreductively cleavable linking group is a disulphide linking group(-S-S-). To determine if a candidate cleavable linking group is asuitable “reductively cleavable linking group,” or for example issuitable for use with a particular antisense polynucleotide agent moietyand particular targeting agent one can look to methods described herein.For example, a candidate can be evaluated by incubation withdithiothreitol (DTT), or other reducing agent using reagents know in theart, which mimic the rate of cleavage which would be observed in a cell,e.g., a target cell. The candidates can also be evaluated underconditions which are selected to mimic blood or serum conditions. Inone, candidate compounds are cleaved by at most about 10% in the blood.In other embodiments, useful candidate compounds are degraded at leastabout 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 timesfaster in the cell (or under in vitro conditions selected to mimicintracellular conditions) as compared to blood (or under in vitroconditions selected to mimic extracellular conditions). The rate ofcleavage of candidate compounds can be determined using standard enzymekinetics assays under conditions chosen to mimic intracellular media andcompared to conditions chosen to mimic extracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In another embodiment, a cleavable linker comprises a phosphate-basedcleavable linking group. A phosphate-based cleavable linking group iscleaved by agents that degrade or hydrolyze the phosphate group. Anexample of an agent that cleaves phosphate groups in cells are enzymessuch as phosphatases in cells. Examples of phosphate-based linkinggroups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—,—S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—,—S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—,—S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S-. Preferred embodimentsare —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—,—O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—,—O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, -S-P(S)(H)—O—,—S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—.These candidates can be evaluated using methods analogous to thosedescribed above.

iii. Acid Cleavable Linking Groups

In another embodiment, a cleavable linker comprises an acid cleavablelinking group. An acid cleavable linking group is a linking group thatis cleaved under acidic conditions. In preferred embodiments acidcleavable linking groups are cleaved in an acidic environment with a pHof about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower),or by agents such as enzymes that can act as a general acid. In a cell,specific low pH organelles, such as endosomes and lysosomes can providea cleaving environment for acid cleavable linking groups. Examples ofacid cleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

iv. Ester-Based Linking Groups

In another embodiment, a cleavable linker comprises an ester-basedcleavable linking group. An ester-based cleavable linking group iscleaved by enzymes such as esterases and amidases in cells. Examples ofester-based cleavable linking groups include but are not limited toesters of alkylene, alkenylene and alkynylene groups. Ester cleavablelinking groups have the general formula —C(O)O—, or —OC(O)—. Thesecandidates can be evaluated using methods analogous to those describedabove.

v. Peptide-Based Cleaving Groups

In yet another embodiment, a cleavable linker comprises a peptide-basedcleavable linking group. A peptide-based cleavable linking group iscleaved by enzymes such as peptidases and proteases in cells.Peptide-based cleavable linking groups are peptide bonds formed betweenamino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)and polypeptides. Peptide-based cleavable groups do not include theamide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynelene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins.The peptide based cleavage group is generally limited to the peptidebond (i.e., the amide bond) formed between amino acids yielding peptidesand proteins and does not include the entire amide functional group.Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the twoadjacent amino acids. These candidates can be evaluated using methodsanalogous to those described above.

In one embodiment, an antisense polynucleotide agent of the invention isconjugated to a carbohydrate through a linker. Non-limiting examples ofantisense polynucleotide agent carbohydrate conjugates with linkers ofthe compositions and methods of the invention include, but are notlimited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention,a ligand is one or more “GalNAc” (N-acetylgalactosamine) derivativesattached through a bivalent or trivalent branched linker.

In one embodiment, a antisense polynucleotide agent of the invention isconjugated to a bivalent or trivalent branched linker selected from thegroup of structures shown in any of formula (XXXII)-(XXXV):

wherein:

q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independentlyfor each occurrence 0-20 and wherein the repeating unit can be the sameor different;

P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(5A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH or CH₂O;

Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherin one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);

R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(Ra)C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) andL^(5C) represent the ligand; i.e. each independently for each occurrencea monosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; andRa is H or aminoacid side chain.Trivalent conjugating GalNAc derivatives areparticularly useful for use with antisense polynucleotide agents forinhibiting the expression of a target gene, such as those of formula(XXXVI):

wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide, such asGalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groupsconjugating GalNAc derivatives include, but are not limited to, thestructures recited above as formulas II, VII, XI, X, and XIII.

Representative U.S. patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; 8,106,022, the entire contents of each of whichare hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within an antisense polynucleotide agent. The presentinvention also includes antisense polynucleotide agents that arechimeric compounds.

“Chimeric” antisense polynucleotide agents or “chimeras,” in the contextof this invention, are antisense polynucleotide agent compounds, whichcontain two or more chemically distinct regions, each made up of atleast one monomer unit, i.e., a nucleotide in the case of an antisensepolynucleotide agent. These antisense polynucleotide agents typicallycontain at least one region wherein the RNA is modified so as to conferupon the antisense polynucleotide agent increased resistance to nucleasedegradation, increased cellular uptake, and/or increased bindingaffinity for the target nucleic acid. An additional region of theantisense polynucleotide agent can serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency of antisensepolynucleotide agent inhibition of gene expression. Consequently,comparable results can often be obtained with shorter antisensepolynucleotide agents when chimeric antisense polynucleotide agents areused, compared to phosphorothioate deoxy antisense polynucleotide agentshybridizing to the same target region. Cleavage of the RNA target can beroutinely detected by gel electrophoresis and, if necessary, associatednucleic acid hybridization techniques known in the art.

In certain instances, the nucleotide of an antisense polynucleotideagent can be modified by a non-ligand group. A number of non-ligandmolecules have been conjugated to antisense polynucleotide agents inorder to enhance the activity, cellular distribution or cellular uptakeof the antisense polynucleotide agent, and procedures for performingsuch conjugations are available in the scientific literature. Suchnon-ligand moieties have included lipid moieties, such as cholesterol(Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61;Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholicacid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), athioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad.Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993,3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992,20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO 1, 1991, 10:111; Kabanov et al., FEBSLett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), aphospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such RNA conjugates havebeen listed above. Typical conjugation protocols involve the synthesisof an RNAs bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction can be performed either with the RNA still bound tothe solid support or following cleavage of the RNA, in solution phase.Purification of the RNA conjugate by HPLC typically affords the pureconjugate.

V. Delivery of an Antisense Polynucleotide Agent of the Invention

The delivery of an antisense polynucleotide agent of the invention to acell e.g., a cell within a subject, such as a human subject (e.g., asubject in need thereof, such as a subject having a LECT2-associateddisease) can be achieved in a number of different ways. For example,delivery may be performed by contacting a cell with an antisensepolynucleotide agent of the invention either in vitro or in vivo. Invivo delivery may also be performed directly by administering acomposition comprising an antisense polynucleotide agent to a subject.

In general, any method of delivering a nucleic acid molecule (in vitroor in vivo) can be adapted for use with an antisense polynucleotideagent of the invention (see e.g., Akhtar S. and Julian R L. (1992)Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporatedherein by reference in their entireties). For in vivo delivery, factorsto consider in order to deliver an antisense polynucleotide agentinclude, for example, biological stability of the delivered molecule,prevention of non-specific effects, and accumulation of the deliveredmolecule in the target tissue. The non-specific effects of an antisensepolynucleotide agent can be minimized by local administration, forexample, by direct injection or implantation into a tissue or topicallyadministering the preparation. Local administration to a treatment sitemaximizes local concentration of the agent, limits the exposure of theagent to systemic tissues that can otherwise be harmed by the agent orthat can degrade the agent, and permits a lower total dose of theantisense polynucleotide agent to be administered. Several studies haveshown successful knockdown of gene products when an antisensepolynucleotide agent is administered locally. For example, intraoculardelivery of a VEGF antisense polynucleotide agent by intravitrealinjection in cynomolgus monkeys (Tolentino, M J., et al (2004) Retina24:132-138) and subretinal injections in mice (Reich, S J., et al (2003)Mol. Vis. 9:210-216) were both shown to prevent neovascularization in anexperimental model of age-related macular degeneration. In addition,direct intratumoral injection of a antisense polynucleotide agent inmice reduces tumor volume (Pille, J., et al (2005) Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J.,et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther.15:515-523). RNA interference has also shown success with local deliveryto the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al(2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A., et al(2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). Foradministering an antisense polynucleotide agent systemically for thetreatment of a disease, the agent can be modified or alternativelydelivered using a drug delivery system; both methods act to prevent therapid degradation of the antisense polynucleotide agent by endo- andexo-nucleases in vivo. Modification of the agent or the pharmaceuticalcarrier can also permit targeting of the antisense polynucleotide agentcomposition to the target tissue and avoid undesirable off-targeteffects. Antisense polynucleotide agent can be modified by chemicalconjugation to lipophilic groups such as cholesterol to enhance cellularuptake and prevent degradation. In an alternative embodiment, theantisense polynucleotide agent can be delivered using drug deliverysystems such as a nanoparticle, a dendrimer, a polymer, liposomes, or acationic delivery system. Positively charged cationic delivery systemsfacilitate binding of an antisense polynucleotide agent molecule(negatively charged) and also enhance interactions at the negativelycharged cell membrane to permit efficient uptake of an antisensepolynucleotide agent by the cell. Cationic lipids, dendrimers, orpolymers can either be bound to an antisense polynucleotide agent, orinduced to form a vesicle or micelle (see e.g., Kim S H., et al (2008)Journal of Controlled Release 129(2):107-116) that encases an antisensepolynucleotide agent. The formation of vesicles or micelles furtherprevents degradation of the antisense polynucleotide agent whenadministered systemically. Methods for making and administeringcationic-antisense polynucleotide agent complexes are well within theabilities of one skilled in the art (see e.g., Sorensen, D R., et al(2003) J. Mol. Biol 327:761-766; Verma, U N, et al (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of antisense polynucleotide agents include DOTAP (Sorensen, DR., et al (2003), supra; Verma, U N., et al (2003), supra),Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S.,et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y., et al (2005)Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Intl. Oncol.26:1087-1091), polyethyleneimine (Bonnet M E., et al (2008) Pharm. Res.Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol.71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm.3:472-487), and polyamidoamines (Tomalia, D A., et al (2007) Biochem.Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804).In some embodiments, an antisense polynucleotide agent forms a complexwith cyclodextrin for systemic administration. Methods foradministration and pharmaceutical compositions of antisensepolynucleotide agents and cyclodextrins can be found in U.S. Pat. No.7,427,605, which is herein incorporated by reference in its entirety.

VI. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions andformulations which include the antisense polynucleotide agents of theinvention. In one embodiment, provided herein are pharmaceuticalcompositions containing an antisense polynucleotide agent, as describedherein, and a pharmaceutically acceptable carrier.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human subjects and animal subjects without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the subject being treated. Some examples ofmaterials which can serve as pharmaceutically-acceptable carriersinclude: (1) sugars, such as lactose, glucose and sucrose; (2) starches,such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)lubricating agents, such as magnesium state, sodium lauryl sulfate andtalc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pHbuffered solutions; (21) polyesters, polycarbonates and/orpolyanhydrides; (22) bulking agents, such as polypeptides and aminoacids (23) serum components, such as serum albumin, HDL and LDL; and(22) other non-toxic compatible substances employed in pharmaceuticalformulations.

The pharmaceutical compositions containing the antisense polynucleotideagents are useful for treating a disease or disorder associated with theexpression or activity of a LECT2 gene, e.g. a LECT2-associated disease.Such pharmaceutical compositions are formulated based on the mode ofdelivery. One example is compositions that are formulated for systemicadministration via parenteral delivery, e.g., by subcutaneous (SC) orintravenous (IV) delivery. Another example is compositions that areformulated for direct delivery into the liver.

The pharmaceutical compositions of the invention may be administered indosages sufficient to inhibit expression of a LECT2 gene. In general, asuitable dose of an anti sense polynucleotide agent of the inventionwill be in the range of about 0.001 to about 200.0 milligrams perkilogram body weight of the recipient per day, generally in the range ofabout 0.1 to 50 mg per kilogram body weight per day. For example, theantisense polynucleotide agent can be administered at about 0.01 mg/kg,about 0.05 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about2 mg/kg, about 3 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg,about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80mg/kg, about 90 mg/kg, or about 100 mg/kg per single dose.

For example, the antisense polynucleotide agent may be administered at adose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 2, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 67, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or about 100mg/kg. Values and ranges intermediate to the recited values are alsointended to be part of this invention.

In another embodiment, the antisense polynucleotide agent isadministered at a dose of about 0.1 to about 100 mg/kg, about 0.25 toabout 100 mg/kg, about 0.5 to about 100 mg/kg, about 0.75 to about 100mg/kg, about 1 to about 100 mg/mg, about 1.5 to about 100 mg/kg, about 2to about 100 mg/kg, about 2.5 to about 100 mg/kg, about 3 to about 100mg/kg, about 3.5 to about 100 mg/kg, about 4 to about 100 mg/kg, about4.5 to about 100 mg/kg, about 5 to about 100 mg/kg, about 7.5 to about100 mg/kg, about 10 to about 100 mg/kg, about 15 to about 100 mg/kg,about 20 to about 100 mg/kg, about 25 to about 100 mg/kg, about 30 toabout 100 mg/kg, about 35 to about 100 mg/kg, about 40 to about 100mg/kg, about 45 to about 100 mg/kg, about 0.1 to about 50 mg/kg, about0.25 to about 50 mg/kg, about 0.5 to about 50 mg/kg, about 0.75 to about50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kb, about2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50mg/kg, about 0.1 to about 45 mg/kg, about 0.25 to about 45 mg/kg, about0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40to about 45 mg/kg, about 0.1 to about 40 mg/kg, about 0.25 to about 40mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40mg/kg, about 0.1 to about 30 mg/kg, about 0.25 to about 30 mg/kg, about0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.1 to about 20mg/kg, about 0.25 to about 20 mg/kg, about 0.5 to about 20 mg/kg, about0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20mg/kg. Values and ranges intermediate to the recited values are alsointended to be part of this invention.

For example, the antisense polynucleotide agent may be administered at adose of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1,9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 2, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 67, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or about 100 mg/kg. Values andranges intermediate to the recited values are also intended to be partof this invention.

In another embodiment, the antisense polynucleotide agent isadministered at a dose of about 0.1 to about 100 mg/kg, about 0.25 toabout 100 mg/kg, about 0.5 to about 100 mg/kg, about 0.75 to about 100mg/kg, about 1 to about 100 mg/mg, about 1.5 to about 100 mg/kg, about 2to about 100 mg/kg, about 2.5 to about 100 mg/kg, about 3 to about 100mg/kg, about 3.5 to about 100 mg/kg, about 4 to about 100 mg/kg, about4.5 to about 100 mg/kg, about 5 to about 100 mg/kg, about 7.5 to about100 mg/kg, about 10 to about 100 mg/kg, about 15 to about 100 mg/kg,about 20 to about 100 mg/kg, about 25 to about 100 mg/kg, about 30 toabout 100 mg/kg, about 35 to about 100 mg/kg, about 40 to about 100mg/kg, about 45 to about 100 mg/kg, about 0.5 to about 50 mg/kg, about0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50mg/kgb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45to about 50 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.5 to about 40mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.5 to about 30mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30mg/kg, about 25 to about 30 mg/kg, about 0.5 to about 20 mg/kg, about0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20mg/kg. In one embodiment, the antisense polynucleotide agent isadministered at a dose of about 0.5 mg/kg to about 10 mg/kg. Values andranges intermediate to the recited values are also intended to be partof this invention.

For example, subjects can be administered, e.g., subcutaneously orintravenously, a single therapeutic amount of antisense polynucleotideagent, such as about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275,0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55,0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825,0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1,9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5,13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5,20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5,27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,67, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, or about 100 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In some embodiments, subjects are administered, e.g., subcutaneously orintravenously, multiple doses of a therapeutic amount of antisensepolynucleotide agent, such as a dose about 0.1, 0.125, 0.15, 0.175, 0.2,0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475,0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75,0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18,18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25,25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 67, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or about 100 mg/kg. Amulti-dose regimine may include administration of a therapeutic amountof antisense polynucleotide agent daily, such as for two days, threedays, four days, five days, six days, seven days, or longer.

In other embodiments, subjects are administered, e.g., subcutaneously orintravenously, a repeat dose of a therapeutic amount of antisensepolynucleotide agent, such as a dose about 0.1, 0.125, 0.15, 0.175, 0.2,0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475,0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75,0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18,18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25,25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 67, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or about 100 mg/kg. Arepeat-dose regimine may include administration of a therapeutic amountof antisense polynucleotide agent on a regular basis, such as everyother day, every third day, every fourth day, twice a week, once a week,every other week, or once a month.

The pharmaceutical composition can be administered by intravenousinfusion over a period of time, such as over a 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, or about a 25minute period. The administration may be repeated, for example, on aregular basis, such as weekly, biweekly (i.e., every two weeks) for onemonth, two months, three months, four months or longer. After an initialtreatment regimen, the treatments can be administered on a less frequentbasis. For example, after administration weekly or biweekly for threemonths, administration can be repeated once per month, for six months ora year or longer.

The pharmaceutical composition can be administered once daily, or theantisense polynucleotide agent can be administered as two, three, ormore sub-doses at appropriate intervals throughout the day or even usingcontinuous infusion or delivery through a controlled releaseformulation. In that case, the antisense polynucleotide agent containedin each sub-dose must be correspondingly smaller in order to achieve thetotal daily dosage. The dosage unit can also be compounded for deliveryover several days, e.g., using a conventional sustained releaseformulation which provides sustained release of the antisensepolynucleotide agent over a several day period. Sustained releaseformulations are well known in the art and are particularly useful fordelivery of agents at a particular site, such as could be used with theagents of the present invention. In this embodiment, the dosage unitcontains a corresponding multiple of the daily dose.

In other embodiments, a single dose of the pharmaceutical compositionscan be long lasting, such that subsequent doses are administered at notmore than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4week intervals. In some embodiments of the invention, a single dose ofthe pharmaceutical compositions of the invention is administered onceper week. In other embodiments of the invention, a single dose of thepharmaceutical compositions of the invention is administered bi-monthly.

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual antisense polynucleotide agentsencompassed by the invention can be made using conventionalmethodologies or on the basis of in vivo testing using an appropriateanimal model, as described elsewhere herein.

A suitable animal model, e.g., a mouse containing a transgene expressinghuman LECT2, can be used to determine the therapeutically effective doseand/or an effective dosage regimen administration of LECT2 antisensepolynucleotide.

The pharmaceutical compositions of the present invention can beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricular,administration.

The antisense polynucleotide agent can be delivered in a manner totarget a particular tissue, such as the liver (e.g., the hepatocytes ofthe liver).

Pharmaceutical compositions and formulations for topical administrationcan include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like can be necessary or desirable. Coated condoms, gloves and thelike can also be useful. Suitable topical formulations include those inwhich the antisense polynucleotide agents featured in the invention arein admixture with a topical delivery agent such as lipids, liposomes,fatty acids, fatty acid esters, steroids, chelating agents andsurfactants. Suitable lipids and liposomes include neutral (e.g.,dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.,dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.,dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). Antisense polynucleotide agents featured in theinvention can be encapsulated within liposomes or can form complexesthereto, in particular to cationic liposomes. Alternatively, antisensepolynucleotide agents can be complexed to lipids, in particular tocationic lipids. Suitable fatty acids and esters include but are notlimited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid,caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid,linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein,dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, anacylcarnitine, an acylcholine, or a C₁₋₂₀ alkyl ester (e.g.,isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceuticallyacceptable salt thereof). Topical formulations are described in detailin U.S. Pat. No. 6,747,014, which is incorporated herein by reference.

A. Antisense Polynucleotide Agent Formulations Comprising MembranousMolecular Assemblies

An anti sense polynucleotide agent for use in the compositions andmethods of the invention can be formulated for delivery in a membranousmolecular assembly, e.g., a liposome or a micelle. As used herein, theterm “liposome” refers to a vesicle composed of amphiphilic lipidsarranged in at least one bilayer, e.g., one bilayer or a plurality ofbilayers. Liposomes include unilamellar and multilamellar vesicles thathave a membrane formed from a lipophilic material and an aqueousinterior. The aqueous portion contains the antisense polynucleotideagent composition. The lipophilic material isolates the aqueous interiorfrom an aqueous exterior, which typically does not include the antisensepolynucleotide agent composition, although in some examples, it may.Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomal bilayer fuses with bilayer of the cellular membranes. Asthe merging of the liposome and cell progresses, the internal aqueouscontents that include the antisense polynucleotide agent are deliveredinto the cell where the antisense polynucleotide agent can specificallybind to a target RNA and can mediate antisense inhibition. In some casesthe liposomes are also specifically targeted, e.g., to direct theantisense polynucleotide agent to particular cell types.

A liposome containing an antisense polynucleotide agent can be preparedby a variety of methods. In one example, the lipid component of aliposome is dissolved in a detergent so that micelles are formed withthe lipid component. For example, the lipid component can be anamphipathic cationic lipid or lipid conjugate. The detergent can have ahigh critical micelle concentration and may be nonionic. Exemplarydetergents include cholate, CHAPS, octylglucoside, deoxycholate, andlauroyl sarcosine. The antisense polynucleotide agent preparation isthen added to the micelles that include the lipid component. Thecationic groups on the lipid interact with the antisense polynucleotideagent and condense around the antisense polynucleotide agent to form aliposome. After condensation, the detergent is removed, e.g., bydialysis, to yield a liposomal preparation of antisense polynucleotideagent.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also be adjusted to favorcondensation.

Methods for producing stable polynucleotide delivery vehicles, whichincorporate a polynucleotide/cationic lipid complex as structuralcomponents of the delivery vehicle, are further described in, e.g., WO96/37194, the entire contents of which are incorporated herein byreference. Liposome formation can also include one or more aspects ofexemplary methods described in Felgner, P. L. etal., Proc. Natl. Acad.Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355; 5,171,678;Bangham, etal. M. Mol. Biol. 23:238, 1965; Olson, et. al. Biochim.Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75:4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, etal. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al.Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipidaggregates of appropriate size for use as delivery vehicles includesonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al.Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be usedwhen consistently small (50 to 200 nm) and relatively uniform aggregatesare desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). Thesemethods are readily adapted to packaging antisense polynucleotide agentpreparations into liposomes.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged nucleicacid molecules to form a stable complex. The positively charged nucleicacid/liposome complex binds to the negatively charged cell surface andis internalized in an endosome. Due to the acidic pH within theendosome, the liposomes are ruptured, releasing their contents into thecell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap nucleicacids rather than complex with it. Since both the nucleic acid and thelipid are similarly charged, repulsion rather than complex formationoccurs. Nevertheless, some nucleic acid is entrapped within the aqueousinterior of these liposomes. pH-sensitive liposomes have been used todeliver nucleic acids encoding the thymidine kinase gene to cellmonolayers in culture. Expression of the exogenous gene was detected inthe target cells (Zhou et al., Journal of Controlled Release, 1992, 19,269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro andin vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO93/24640; WO 91/16024; Feigner, J. Biol. Chem. 269:2550, 1994; Nabel,Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649,1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporine A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4(6) 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

In one embodiment, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver antisense polynucleotide agents to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated antisense polynucleotide agents in their internalcompartments from metabolism and degradation (Rosoff, in “PharmaceuticalDosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p.245). Important considerations in the preparation of liposomeformulations are the lipid surface charge, vesicle size and the aqueousvolume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of Antisense polynucleotideagent (see, e.g., Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMAand its use with DNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.)is an effective agent for the delivery of highly anionic nucleic acidsinto living tissue culture cells that comprise positively charged DOTMAliposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (see, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta1065:8, 1991). For certain cell lines, these liposomes containingconjugated cationic lipids, are said to exhibit lower toxicity andprovide more efficient transfection than the DOTMA-containingcompositions. Other commercially available cationic lipid productsinclude DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine(DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationiclipids suitable for the delivery of oligonucleotides are described in WO98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topicaladministration; liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer an antisense polynucleotide agent into the skin.In some implementations, liposomes are used for delivering antisensepolynucleotide agenst to epidermal cells and also to enhance thepenetration of antisense polynucleotide agenst into dermal tissues,e.g., into skin. For example, the liposomes can be applied topically.Topical delivery of drugs formulated as liposomes to the skin has beendocumented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992,vol. 2,405-410 and du Plessis et al., Antiviral Research, 18, 1992,259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690,1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth.Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth.Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad.Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith antisense polynucleotide agents are useful for treating adermatological disorder.

Liposomes that include antisense polynucleotide agent can be made highlydeformable. Such deformability can enable the liposomes to penetratethrough pore that are smaller than the average radius of the liposome.For example, transfersomes are a type of deformable liposomes.Transferosomes can be made by adding surface edge activators, usuallysurfactants, to a standard liposomal composition. Transfersomes thatinclude antisense polynucleotide agents can be delivered, for example,subcutaneously by infection in order to deliver antisense polynucleotideagents to keratinocytes in the skin. In order to cross intact mammalianskin, lipid vesicles must pass through a series of fine pores, each witha diameter less than 50 nm, under the influence of a suitabletransdermal gradient. In addition, due to the lipid properties, thesetransferosomes can be self-optimizing (adaptive to the shape of pores,e.g., in the skin), self-repairing, and can frequently reach theirtargets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described inU.S. provisional application Ser. Nos. 61/018,616, filed Jan. 2, 2008;61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008;61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCTapplication no PCT/US2007/080331, filed Oct. 3, 2007 also describesformulations that are amenable to the present invention.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes can be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, in“Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, N.Y.,1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in “Pharmaceutical Dosage Forms”, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

The antisense polynucleotide agent for use in the compositions andmethods of the invention can also be provided as micellar formulations.“Micelles” are defined herein as a particular type of molecular assemblyin which amphipathic molecules are arranged in a spherical structuresuch that all the hydrophobic portions of the molecules are directedinward, leaving the hydrophilic portions in contact with the surroundingaqueous phase. The converse arrangement exists if the environment ishydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of the antisensepolynucleotide agent composition, an alkali metal C₈ to C₂₂ alkylsulphate, and a micelle forming compounds. Exemplary micelle formingcompounds include lecithin, hyaluronic acid, pharmaceutically acceptablesalts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract,cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein,monooleates, monolaurates, borage oil, evening of primrose oil, menthol,trihydroxy oxo cholanyl glycine and pharmaceutically acceptable saltsthereof, glycerin, polyglycerin, lysine, polylysine, triolein,polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethersand analogues thereof, chenodeoxycholate, deoxycholate, and mixturesthereof. The micelle forming compounds may be added at the same time orafter addition of the alkali metal alkyl sulphate. Mixed micelles willform with substantially any kind of mixing of the ingredients butvigorous mixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which containsthe antisense polynucleotide agent composition and at least the alkalimetal alkyl sulphate. The first micellar composition is then mixed withat least three micelle forming compounds to form a mixed micellarcomposition. In another method, the micellar composition is prepared bymixing the antisense polynucleotide agent composition, the alkali metalalkyl sulphate and at least one of the micelle forming compounds,followed by addition of the remaining micelle forming compounds, withvigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol and/or m-cresol may be added with the micelleforming ingredients. An isotonic agent such as glycerin may also beadded after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Incertain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g., atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

B. Lipid Particles

Antisense polynucleotide agents of in the invention may be fullyencapsulated in a lipid formulation, e.g., a LNP, or other nucleicacid-lipid particle.

As used herein, the term “LNP” refers to a stable nucleic acid-lipidparticle comprising a lipid layer encapsulating a pharmaceuticallyactive molecule. LNPs typically contain a cationic lipid, a non-cationiclipid, and a lipid that prevents aggregation of the particle (e.g., aPEG-lipid conjugate). LNPs are extremely useful for systemicapplications, as they exhibit extended circulation lifetimes followingintravenous (i.v.) injection and accumulate at distal sites (e.g., sitesphysically separated from the administration site). LNPs include“pSPLP,” which include an encapsulated condensing agent-nucleic acidcomplex as set forth in PCT Publication No. WO 00/03683. The particlesof the present invention typically have a mean diameter of about 50 nmto about 150 nm, more typically about 60 nm to about 130 nm, moretypically about 70 nm to about 110 nm, most typically about 70 nm toabout 90 nm, and are substantially nontoxic. In addition, the nucleicacids when present in the nucleic acid-lipid particles of the presentinvention are resistant in aqueous solution to degradation with anuclease. Nucleic acid-lipid particles and their method of preparationare disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484;6,586,410; 6,815,432; 6,858,225; 8,158,601; and 8,058,069; U.S.Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to antisense polynucleotide agent ratio) will be in the range offrom about 1:1 to about 50:1, from about 1:1 to about 25:1, from about3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about9:1, or about 6:1 to about 9:1. Ranges intermediate to the above recitedranges are also contemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(I -(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(I -(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.C1),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6a5)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxo1-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech G1), or a mixture thereof. The cationic lipid can comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

In another embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane can be used toprepare lipid-santisense polynucleotide agent nanoparticles. Synthesisof 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described inU.S. provisional patent application No. 61/107,998 filed on Oct. 23,2008, which is herein incorporated by reference.

In one embodiment, the lipid-antisense polynucleotide agent particleincludes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10%DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particlesize of 63.0±20 nm and a 0.027 antisense polynucleotide agent/LipidRatio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutrallipid including, but not limited to, di stearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1 -trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid can be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (Ci₈). The conjugated lipid that preventsaggregation of particles can be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

In one embodiment, the lipidoid ND98.4HC1 (MW 1487) (see U .S. patentapplication Ser. No. 12/056,230, filed March 26, 2008, which isincorporated herein by reference), Cholesterol (Sigma-Aldrich), andPEG-Ceramide C16 (Avanti Polar Lipids) can be used to preparelipid-antisense polynucleotide agent nanoparticles (i.e., LNP01particles). Stock solutions of each in ethanol can be prepared asfollows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions canthen be combined in a, e.g., 42:48:10 molar ratio. The combined lipidsolution can be mixed with aqueous antisense polynucleotide agent (e.g.,in sodium acetate pH 5) such that the final ethanol concentration isabout 35-45% and the final sodium acetate concentration is about 100-300mM. Lipid-antisense polynucleotide agent nanoparticles typically formspontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture can be extruded througha polycarbonate membrane (e.g., 100 nm cut-off) using, for example, athermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). Insome cases, the extrusion step can be omitted. Ethanol removal andsimultaneous buffer exchange can be accomplished by, for example,dialysis or tangential flow filtration. Buffer can be exchanged with,for example, phosphate buffered saline (PBS) at about pH 7, e.g., aboutpH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or aboutpH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-antisense polynucleotide agent formulationsare described in

TABLE 1 Table 1 cationic lipid/non-cationic lipid/cholesterol/PEG-lipidconjugate Ionizable/Cationic Lipid Lipid:santisense polynucleotide agentratio SNALP-1 1,2-Dilinolenyloxy-N,N-dimethylaminopropaneDLinDMA/DPPC/Cholesterol/PEG-cDMA (DLinDMA) (57.1/7.1/34.4/1.4)lipid:santisense polynucleotide agent ~7:1 2-XTC2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DPPC/Cholesterol/PEG-cDMAdioxolane (XTC) 57.1/7.1/34.4/1.4 lipid:santisense polynucleotide agent~7:1 LNP05 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5lipid:santisense polynucleotide agent ~6:1 LNP062,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:santisense polynucleotide agent~11:1 LNP07 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5,lipid:santisense polynucleotide agent ~6:1 LNP082,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMGdioxolane (XTC) 60/7.5/31/1.5, lipid:santisense polynucleotide agent~11:1 LNP09 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 50/10/38.5/1.5Lipid:santisense polynucleotide agent 10:1 LNP10(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-ALN100/DSPC/Cholesterol/PEG-DMG octadeca-9,12-dienyl)tetrahydro-3aH-50/10/38.5/1.5 cyclopenta[d][1,3]dioxol-5-amine (ALN100)Lipid:santisense polynucleotide agent 10:1 LNP11(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- MC-3/DSPC/Cholesterol/PEG-DMGtetraen-19-yl 4-(dimethylamino)butanoate 50/10/38.5/1.5 (MC3)Lipid:santisense polynucleotide agent 10:1 LNP121,1′-(2-(4-(2-((2-(bis(2- Tech G1/DSPC/Cholesterol/PEG-DMGhydroxydodecyl)amino)ethyl)(2- 50/10/38.5/1.5hydroxydodecyl)amino)ethyl)piperazin-1- Lipid:santisense polynucleotideagent 10:1 yl)ethylazanediyl)didodecan-2-ol (Tech G1) LNP13 XTCXTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:santisense polynucleotideagent: 33:1 LNP14 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:santisensepolynucleotide agent: 11:1 LNP15 MC3MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:santisensepolynucleotide agent: 11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG50/10/38.5/1.5 Lipid:santisense polynucleotide agent: 7:1 LNP17 MC3MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:santisense polynucleotideagent: 10:1 LNP18 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5Lipid:santisense polynucleotide agent: 12:1 LNP19 MC3MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:santisense polynucleotide agent:8:1 LNP20 MC3 MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:santisensepolynucleotide agent: 10:1 LNP21 C12-200 C12-200/DSPC/Chol/PEG-DSG50/10/38.5/1.5 Lipid:santisense polynucleotide agent: 7:1 LNP22 XTCXTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:santisense polynucleotideagent: 10:1 DSPC: distearoylphosphatidylcholine DPPC:dipalmitoylphosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol(C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000) PEG-DSG:PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of2000) PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avgmol wt of 2000) SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane(DLinDMA)) comprising formulations are described in InternationalPublication No. WO2009/127060, filed Apr. 15, 2009, which is herebyincorporated by reference.

XTC comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No.61/156,851, filed Mar. 2, 2009; U.S. Provisional Ser. No. filed Jun. 10,2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S.Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and InternationalApplication No. PCT/US2010/022614, filed Jan. 29, 2010, which are herebyincorporated by reference.

MC3 comprising formulations are described, e.g., in U.S. Publication No.2010/0324120, filed Jun. 10, 2010, the entire contents of which arehereby incorporated by reference.

ALNY-100 comprising formulations are described, e.g., Internationalpatent application number PCT/US09/63933, filed on Nov. 10, 2009, whichis hereby incorporated by reference.

C12-200 comprising formulations are described in U.S. Provisional Ser.No. 61/175,770, filed May 5, 2009 and International Application No.PCT/US10/33777, filed May 5, 2010, which are hereby incorporated byreference.

Synthesis of Ionizable/Cationic Lipids

Any of the compounds, e.g., cationic lipids and the like, used in thenucleic acid-lipid particles of the invention can be prepared by knownorganic synthesis techniques, including the methods described in moredetail in the Examples. All substituents are as defined below unlessindicated otherwise.

“Alkyl” means a straight chain or branched, noncyclic or cyclic,saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturatedbranched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl,isopentyl, and the like. Representative saturated cyclic alkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; whileunsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, andthe like.

“Alkenyl” means an alkyl, as defined above, containing at least onedouble bond between adjacent carbon atoms. Alkenyls include both cis andtrans isomers. Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-l-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, whichadditionally contains at least one triple bond between adjacent carbons.Representative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at thepoint of attachment is substituted with an oxo group, as defined below.For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acylgroups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, oraromatic, and which contains from 1 or 2 heteroatoms independentlyselected from nitrogen, oxygen and sulfur, and wherein the nitrogen andsulfur heteroatoms can be optionally oxidized, and the nitrogenheteroatom can be optionally quaternized, including bicyclic rings inwhich any of the above heterocycles are fused to a benzene ring. Theheterocycle can be attached via any heteroatom or carbon atom.Heterocycles include heteroaryls as defined below. Heterocycles includemorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl,hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substitutedalkenyl”, “optionally substituted alkynyl”, “optionally substitutedacyl”, and “optionally substituted heterocycle” means that, whensubstituted, at least one hydrogen atom is replaced with a substituent.In the case of an oxo substituent (═O) two hydrogen atoms are replaced.In this regard, substituents include oxo, halogen, heterocycle, —CN,—ORx, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy,—SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same ordifferent and independently hydrogen, alkyl or heterocycle, and each ofsaid alkyl and heterocycle substituents can be further substituted withone or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy,—NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and—SOnNRxRy.

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, protecting groups can be used. Protecting groupmethodology is well known to those skilled in the art (see, for example,Protective Groups in Organic Synthesis, Green, T. W. et al.,Wiley-Interscience, New York City, 1999). Briefly, protecting groupswithin the context of this invention are any group that reduces oreliminates unwanted reactivity of a functional group. A protecting groupcan be added to a functional group to mask its reactivity during certainreactions and then removed to reveal the original functional group. Insome embodiments an “alcohol protecting group” is used. An “alcoholprotecting group” is any group which decreases or eliminates unwantedreactivity of an alcohol functional group. Protecting groups can beadded and removed using techniques well known in the art.

Synthesis of Formula A

In some embodiments, nucleic acid-lipid particles of the invention areformulated using a cationic lipid of formula A:

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can beoptionally substituted, and R3 and R4 are independently lower alkyl orR3 and R4 can be taken together to form an optionally substitutedheterocyclic ring. In some embodiments, the cationic lipid is XTC(2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, thelipid of formula A above can be made by the following Reaction Schemes 1or 2, wherein all substituents are as defined above unless indicatedotherwise.

Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl,each can be optionally substituted, and R3 and R4 are independentlylower alkyl or R3 and R4 can be taken together to form an optionallysubstituted heterocyclic ring, can be prepared according to Scheme 1.Ketone 1 and bromide 2 can be purchased or prepared according to methodsknown to those of ordinary skill in the art. Reaction of 1 and 2 yieldsketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A.The lipids of formula A can be converted to the corresponding ammoniumsalt with an organic salt of formula 5, where X is anion counter ionselected from halogen, hydroxide, phosphate, sulfate, or the like.

Alternatively, the ketone 1 starting material can be prepared accordingto Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased orprepared according to methods known to those of ordinary skill in theart. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to thecorresponding lipids of formula A is as described in Scheme 1.

Synthesis ofMC3

Preparation of DLin-M-C3 -DMA (i.e.,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate) was as follows. A solution of(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g),4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g),4-N,N-dimethylaminopyridine (0.61g) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) indichloromethane (5 mL) was stirred at room temperature overnight. Thesolution was washed with dilute hydrochloric acid followed by diluteaqueous sodium bicarbonate. The organic fractions were dried overanhydrous magnesium sulphate, filtered and the solvent removed on arotovap. The residue was passed down a silica gel column (20 g) using a1-5% methanol/dichloromethane elution gradient. Fractions containing thepurified product were combined and the solvent removed, yielding acolorless oil (0.54 g).

Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] was performed using the followingscheme 3:

Synthesis of 515

To a stirred suspension of LiA1H4 (3.74 g, 0.09852 mol) in 200 mlanhydrous THF in a two neck RBF (1L), was added a solution of 514 (10g,0.04926mo1) in 70 mL of THF slowly at 0° C. under nitrogen atmosphere.After complete addition, reaction mixture was warmed to room temperatureand then heated to reflux for 4 h. Progress of the reaction wasmonitored by TLC. After completion of reaction (by TLC) the mixture wascooled to 0° C. and quenched with careful addition of saturated Na2SO4solution. Reaction mixture was stirred for 4 h at room temperature andfiltered off. Residue was washed well with THF. The filtrate andwashings were mixed and diluted with 400 mL dioxane and 26 mL conc. HC1and stirred for 20 minutes at room temperature. The volatilities werestripped off under vacuum to furnish the hydrochloride salt of 515 as awhite solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz): δ=9.34 (broad, 2H),5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).

Synthesis of 516

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL twoneck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. undernitrogen atmosphere. After a slow addition ofN-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dryDCM, reaction mixture was allowed to warm to room temperature. Aftercompletion of the reaction (2-3 h by TLC) mixture was washedsuccessively with 1N HCl solution (1×100 mL) and saturated NaHCO3solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4and the solvent was evaporated to give crude material which was purifiedby silica gel column chromatography to get 516 as sticky mass. Yield:llg (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27(m, 5H), 5.69 (s, 2H),5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60(m, 2H), 2.30-2.25(m,2H). LC-MS [M+H]−232.3 (96.94%).

Synthesis of 517A and 517E

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of220 mL acetone and water (10:1) in a single neck 500 mL RBF and to itwas added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanolat room temperature. After completion of the reaction (˜3 h), themixture was quenched with addition of solid Na2SO3 and resulting mixturewas stirred for 1.5 h at room temperature. Reaction mixture was dilutedwith DCM (300 mL) and washed with water (2×100 mL) followed by saturatedNaHCO3 (1×50 mL) solution, water (1×30 mL) and finally with brine (1×50mL). Organic phase was dried over an.Na2SO4 and solvent was removed invacuum. Silica gel column chromatographic purification of the crudematerial was afforded a mixture of diastereomers, which were separatedby prep HPLC. Yield: −6 g crude

517A -Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz):δ=7.39-7.31(m, 5H), 5.04(s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47(d, 2H),3.94-3.93(m, 2H), 2.71(s, 3H), 1.72-1.67(m, 4H). LC-MS -[M+H]-266.3,[M+NH4 +]-283.5 present, HPLC-97.86%. Stereochemistry confirmed byX-ray.

Synthesis of 518

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil.1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33(m, 4H), 7.30-7.27(m, 1H),5.37-5.27(m, 8H), 5.12(s, 2H), 4.75(m,1H), 4.58-4.57(m,2H),2.78-2.74(m,7H), 2.06-2.00(m,8H), 1.96-1.91(m, 2H), 1.62(m, 4H), 1.48(m,2H), 1.37-1.25(br m, 36H), 0.87(m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519

A solution of compound 518 (1 eq) in hexane (15 mL) was added in adrop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq).After complete addition, the mixture was heated at 40° C. over 0.5 hthen cooled again on an ice bath. The mixture was carefully hydrolyzedwith saturated aqueous Na2SO4 then filtered through celite and reducedto an oil. Column chromatography provided the pure 519 (1.3 g, 68%)which was obtained as a colorless oil. 13C NMR δ=130.2, 130.1 (x2),127.9 (x3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (x2), 29.7,29.6 (x2), 29.5 (x3), 29.3 (x2), 27.2 (x3), 25.6, 24.5, 23.3, 226, 14.1;Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+Calc. 654.6,Found 654.6.

Formulations prepared by either the standard or extrusion-free methodcan be characterized in similar manners. For example, formulations aretypically characterized by visual inspection. They should be whitishtranslucent solutions free from aggregates or sediment. Particle sizeand particle size distribution of lipid-nanoparticles can be measured bylight scattering using, for example, a Malvern Zetasizer Nano ZS(Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nmin size. The particle size distribution should be unimodal. The totalantisense polynucleotide agent concentration in the formulation, as wellas the entrapped fraction, is estimated using a dye exclusion assay. Asample of the formulated antisense polynucleotide agent can be incubatedwith an RNA-binding dye, such as Ribogreen (Molecular Probes) in thepresence or absence of a formulation disrupting surfactant, e.g., 0.5%Triton-X100. The total antisense polynucleotide agent in the formulationcan be determined by the signal from the sample containing thesurfactant, relative to a standard curve. The entrapped fraction isdetermined by subtracting the “free” antisense polynucleotide agentcontent (as measured by the signal in the absence of surfactant) fromthe total antisense polynucleotide agent content. Percent entrappedantisense polynucleotide agent is typically >85%. For SNALP formulation,the particle size is at least 30 nm, at least 40 nm, at least 50 nm, atleast 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least100 nm, at least 110 nm, and at least 120 nm. The suitable range istypically about at least 50 nm to about at least 110 nm, about at least60 nm to about at least 100 nm, or about at least 80 nm to about atleast 90 nm.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders can be desirable. In some embodiments, oralformulations are those in which the anti sense polynucleotide agentsfeatured in the invention are administered in conjunction with one ormore penetration enhancer surfactants and chelators. Suitablesurfactants include fatty acids and/or esters or salts thereof, bileacids and/or salts thereof. Suitable bile acids/salts includechenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA),cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid,glycholic acid, glycodeoxycholic acid, taurocholic acid,taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodiumglycodihydrofusidate. Suitable fatty acids include arachidonic acid,undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid,myristic acid, palmitic acid, stearic acid, linoleic acid, linolenicacid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, anacylcholine, or a monoglyceride, a diglyceride or a pharmaceuticallyacceptable salt thereof (e.g., sodium). In some embodiments,combinations of penetration enhancers are used, for example, fattyacids/salts in combination with bile acids/salts. One exemplarycombination is the sodium salt of lauric acid, capric acid and UDCA.Further penetration enhancers include polyoxyethylene-9-lauryl ether,polyoxyethylene-20-cetyl ether. Antisense polynucleotide agents featuredin the invention can be delivered orally, in granular form includingsprayed dried particles, or complexed to form micro or nanoparticles.Antisense polynucleotide agent complexing agents include poly-aminoacids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor antisense polynucleotide agents and their preparation are describedin detail in U.S. Patent 6,887,906, US Publn. No. 20030027780, and U.S.Pat. No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration caninclude sterile aqueous solutions which can also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions can be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which canconveniently be presented in unit dosage form, can be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention can be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention can also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions can further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

C. Additional Formulations

i. Emulsions

The compositions of the present invention can be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions can be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions can contain additional componentsin addition to the dispersed phases, and the active drug which can bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants can also be present in emulsions asneeded. Pharmaceutical emulsions can also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion can be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatcan be incorporated into either phase of the emulsion. Emulsifiers canbroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, L V., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants can beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, NY Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tri stearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that can readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used can be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff,in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

ii. Microemulsions

In one embodiment of the present invention, the compositions ofantisense polynucleotide agents are formulated as microemulsions. Amicroemulsion can be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and DrugDelivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typicallymicroemulsions are systems that are prepared by first dispersing an oilin an aqueous surfactant solution and then adding a sufficient amount ofa fourth component, generally an intermediate chain-length alcohol toform a transparent system. Therefore, microemulsions have also beendescribed as thermodynamically stable, isotropically clear dispersionsof two immiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (P0310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (S0750), decaglycerol decaoleate (DA0750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions can, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase can typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase can include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U .S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions can form spontaneouslywhen their components are brought together at ambient temperature. Thiscan be particularly advantageous when formulating thermolabile drugs,peptides or antisense polynucleotide agents. Microemulsions have alsobeen effective in the transdermal delivery of active components in bothcosmetic and pharmaceutical applications. It is expected that themicroemulsion compositions and formulations of the present inventionwill facilitate the increased systemic absorption of anti sensepolynucleotide agents from the gastrointestinal tract, as well asimprove the local cellular uptake of antisense polynucleotide agents andnucleic acids.

Microemulsions of the present invention can also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the antisensepolynucleotide agents of the present invention. Penetration enhancersused in the microemulsions of the present invention can be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

iii. Microparticles

An antisense polynucleotide agent of the invention may be incorporatedinto a particle, e.g., a microparticle. Microparticles can be producedby spray-drying, but may also be produced by other methods includinglyophilization, evaporation, fluid bed drying, vacuum drying, or acombination of these techniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly antisense polynucleotide agents, to the skin of animals.Most drugs are present in solution in both ionized and nonionized forms.However, usually only lipid soluble or lipophilic drugs readily crosscell membranes. It has been discovered that even non-lipophilic drugscan cross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

Penetration enhancers can be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p.92). Each of the above mentioned classes ofpenetration enhancers are described below in greater detail.

Surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of antisensepolynucleotide agents through the mucosa is enhanced. In addition tobile salts and fatty acids, these penetration enhancers include, forexample, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether andpolyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants andpolymers in drug delivery, Informa Health Care, New York, N.Y., 2002;Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid(n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₂₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g.,Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers,Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (see e.g., Malmsten,M. Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds.,McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts,and their synthetic derivatives, act as penetration enhancers. Thus theterm “bile salts” includes any of the naturally occurring components ofbile as well as any of their synthetic derivatives. Suitable bile saltsinclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present invention, canbe defined as compounds that remove metallic ions from solution byforming complexes therewith, with the result that absorption ofantisense polynucleotide agents through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents includebut are not limited to disodium ethylenediaminetetraacetate (EDTA),citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylateand homovanilate), N-acyl derivatives of collagen, laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A. etal., Excipient development for pharmaceutical, biotechnology, and drugdelivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al.,J. Control Rel., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancingcompounds can be defined as compounds that demonstrate insignificantactivity as chelating agents or as surfactants but that nonethelessenhance absorption of antisense polynucleotide agents through thealimentary mucosa (see e.g., Muranishi, Critical Reviews in TherapeuticDrug Carrier Systems, 1990, 7, 1-33). This class of penetrationenhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39,621-626).

Agents that enhance uptake of antisense polynucleotide agents at thecellular level can also be added to the pharmaceutical and othercompositions of the present invention. For example, cationic lipids,such as lipofectin (Junichi et al, U .S. Pat. No. 5,705,188), cationicglycerol derivatives, and polycationic molecules, such as polylysine(Lollo et al., PCT Application WO 97/30731), are also known to enhancethe cellular uptake of antisense polynucleotide agents. Examples ofcommercially available transfection reagents include, for exampleLipofectamine™ (Invitrogen; Carlsbad, Calif.), Lipofectamine 2000™(Invitrogen; Carlsbad, Calif.), 293fectin™ (Invitrogen; Carlsbad,Calif.), Cellfectin™ (Invitrogen; Carlsbad, Calif.), DMRIE-C™(Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX (Invitrogen; Carlsbad,Calif.), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, Calif.),Lipofectamine™ (Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen;Carlsbad, Calif.), Oligofectamine™ (Invitrogen; Carlsbad, Calif.),Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 TransfectionReagent (Roche; Grenzacherstrasse, Switzerland), DOTAP LiposomalTransfection Reagent (Grenzacherstrasse, Switzerland), DOSPER LiposomalTransfection Reagent (Grenzacherstrasse, Switzerland), or Fugene(Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison,Wis.), Tfx™-20 Reagent (Promega; Madison, WI), Tfx™-50 Reagent (Promega;Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille, France),EcoTransfect (OZ Biosciences; Marseille, France), TransPass^(a) D1Transfection Reagent (New England Biolabs; Ipswich, Mass., USA),LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectinTransfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTERTransfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2Transfection reagent (Genlantis; San Diego, Calif., USA), CytofectinTransfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect(Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA),UniFECTOR (B-Bridge International; Mountain View, Calif., USA),SureFECTOR (B-Bridge International; Mountain View, Calif., USA), orHiFect™ (B-Bridge International, Mountain View, Calif., USA), amongothers.

Other agents can be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

v. Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioated antisense polynucleotide agent in hepatic tissue canbe reduced when it is coadministered with polyinosinic acid, dextransulfate, polycytidic acid or4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense polynucleotide agent Res. Dev., 1995, 5, 115-121; Takakura etal., Antisense polynucleotide agent & Nucl. Acid Drug Dev., 1996, 6,177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient can be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions can also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

vii. Other Components

The compositions of the present invention can additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions can contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or can contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more antisense polynucleotide agents and(b) one or more biologic agents which function by a non-antisensepolynucleotide mechanism. Examples of such biologic agents includeagents that interfere with an interaction of LECT2 and at least oneLECT2 binding partner. Toxicity and therapeutic efficacy of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD_(50/)ED₅₀. Compounds that exhibit hightherapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein in the invention lies generally within arange of circulating concentrations that include the EDso with little orno toxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC₅₀ (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the antisensepolynucleotide agents featured in the invention can be administered incombination with other known agents effective in treatment ofpathological processes mediated by LECT2 gene expression. In any event,the administering physician can adjust the amount and timing ofantisense polynucleotide agent administration on the basis of resultsobserved using standard measures of efficacy known in the art ordescribed herein.

VII. Methods For Inhibiting LECT2 Gene Expression

The present invention provides methods of inhibiting expression of LECT2in a cell. The methods include contacting a cell with an antisensepolynucleotide agent of the invention in an amount effective to inhibitexpression of the LECT2 gene in the cell, thereby inhibiting expressionof the LECT2 in the cell.

Contacting of a cell with an antisense polynucleotide agent may be donein vitro or in vivo. Contacting a cell in vivo with the antisensepolynucleotide agent includes contacting a cell or group of cells withina subject, e.g., a human subject, with the antisense polynucleotideagent. Combinations of in vitro and in vivo methods of contacting arealso possible. Contacting may be direct or indirect, as discussed above.Furthermore, contacting a cell may be accomplished via a targetingligand, including any ligand described herein or known in the art. Inpreferred embodiments, the targeting ligand is a carbohydrate moiety,e.g., a GalNAc₃ ligand, or any other ligand that directs the antisensepolynucleotide agent to a site of interest, e.g., the liver of asubject.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating” and other similar terms, andincludes any level of inhibition.

The phrase “inhibiting expression of a LECT2” is intended to refer toinhibition of expression of any LECT2 gene (such as, e.g., a mouse LECT2gene, a rat LECT2 gene, a monkey LECT2 gene, or a human LECT2 gene) aswell as variants or mutants of a LECT2 gene. Thus, the LECT2 gene may bea wild-type LECT2 gene, a mutant LECT2 gene, or a transgenic LECT2 genein the context of a genetically manipulated cell, group of cells, ororganism.

“Inhibiting expression of a LECT2 gene” includes any level of inhibitionof a LECT2 gene, e.g., at least partial suppression of the expression ofa LECT2 gene. The expression of the LECT2 gene may be assessed based onthe level, or the change in the level, of any variable associated withLECT2 gene expression, e.g., LECT2 mRNA level, LECT2 protein level. Thislevel may be assessed in an individual cell or in a group of cells,including, for example, a sample derived from a subject.

Inhibition may be assessed by a decrease in an absolute or relativelevel of one or more variables that are associated with LECT2 expressioncompared with a control level. The control level may be any type ofcontrol level that is utilized in the art, e.g., a pre-dose baselinelevel, or a level determined from a similar subject, cell, or samplethat is untreated or treated with a control (such as, e.g., buffer onlycontrol or inactive agent control).

In some embodiments of the methods of the invention, expression of aLECT2 gene is inhibited by at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%. at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at leastabout 99%.

Inhibition of the expression of a LECT2 gene may be manifested by areduction of the amount of mRNA expressed by a first cell or group ofcells (such cells may be present, for example, in a sample derived froma subject) in which a LECT2 gene is transcribed and which has or havebeen treated (e.g., by contacting the cell or cells with an antisensepolynucleotide agent of the invention, or by administering an antisensepolynucleotide agent of the invention to a subject in which the cellsare or were present) such that the expression of a LECT2 gene isinhibited, as compared to a second cell or group of cells substantiallyidentical to the first cell or group of cells but which has not or havenot been so treated (control cell(s)). In preferred embodiments, theinhibition is assessed by expressing the level of mRNA in treated cellsas a percentage of the level of mRNA in control cells, using thefollowing formula:

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

Alternatively, inhibition of the expression of a LECT2 gene may beassessed in terms of a reduction of a parameter that is functionallylinked to LECT2 gene expression, e.g., LECT2 protein expression. LECT2gene silencing may be determined in any cell expressing LECT2, eitherconstitutively or by genomic engineering, and by any assay known in theart.

Inhibition of the expression of a LECT2 protein may be manifested by areduction in the level of the LECT2 protein that is expressed by a cellor group of cells (e.g., the level of protein expressed in a samplederived from a subject). As explained above for the assessment of mRNAsuppression, the inhibiton of protein expression levels in a treatedcell or group of cells may similarly be expressed as a percentage of thelevel of protein in a control cell or group of cells.

A control cell or group of cells that may be used to assess theinhibition of the expression of a LECT2 gene includes a cell or group ofcells that has not yet been contacted with an antisense polynucleotideagent of the invention. For example, the control cell or group of cellsmay be derived from an individual subject (e.g., a human or animalsubject) prior to treatment of the subject with an antisensepolynucleotide agent.

The level of LECT2 mRNA that is expressed by a cell or group of cellsmay be determined using any method known in the art for assessing mRNAexpression. In one embodiment, the level of expression of LECT2 in asample is determined by detecting a transcribed polynucleotide, orportion thereof, e.g., mRNA of the LECT2 gene. RNA may be extracted fromcells using RNA extraction techniques including, for example, using acidphenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis),RNeasy RNA preparation kits (Qiagen) or PAXgene (PreAnalytix,Switzerland). Typical assay formats utilizing ribonucleic acidhybridization include nuclear run-on assays, RT-PCR, RNase protectionassays (Melton et al., Nuc. Acids Res. 12:7035), Northern blotting, insitu hybridization, and microarray analysis.

In one embodiment, the level of expression of LECT2 is determined usinga nucleic acid probe. The term “probe”, as used herein, refers to anymolecule that is capable of selectively binding to a specific LECT2.Probes can be synthesized by one of skill in the art, or derived fromappropriate biological preparations. Probes may be specifically designedto be labeled. Examples of molecules that can be utilized as probesinclude, but are not limited to, RNA, DNA, proteins, antibodies, andorganic molecules.

Isolated mRNA can be used in hybridization or amplification assays thatinclude, but are not limited to, Southern or Northern analyses,polymerase chain reaction (PCR) analyses and probe arrays. One methodfor the determination of mRNA levels involves contacting the isolatedmRNA with a nucleic acid molecule (probe) that can hybridize to LECT2mRNA. In one embodiment, the mRNA is immobilized on a solid surface andcontacted with a probe, for example by running the isolated mRNA on anagarose gel and transferring the mRNA from the gel to a membrane, suchas nitrocellulose. In an alternative embodiment, the probe(s) areimmobilized on a solid surface and the mRNA is contacted with theprobe(s), for example, in an Affymetrix gene chip array. A skilledartisan can readily adapt known mRNA detection methods for use indetermining the level of LECT2 mRNA.

An alternative method for determining the level of expression of LECT2in a sample involves the process of nucleic acid amplification and/orreverse transcriptase (to prepare cDNA) of for example mRNA in thesample, e.g., by RT-PCR (the experimental embodiment set forth inMullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany(1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardiet al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplificationmethod, followed by the detection of the amplified molecules usingtechniques well known to those of skill in the art. These detectionschemes are especially useful for the detection of nucleic acidmolecules if such molecules are present in very low numbers. Inparticular aspects of the invention, the level of expression of LECT2 isdetermined by quantitative fluorogenic RT-PCR (i.e., the TaqMan™System).

The expression levels of LECT2 mRNA may be monitored using a membraneblot (such as used in hybridization analysis such as Northern, Southern,dot, and the like), or microwells, sample tubes, gels, beads or fibers(or any solid support comprising bound nucleic acids). See U.S. Pat.Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which areincorporated herein by reference. The determination of LECT2 expressionlevel may also comprise using nucleic acid probes in solution.

In preferred embodiments, the level of mRNA expression is assessed usingbranched DNA (bDNA) assays or real time PCR (qPCR). The use of thesemethods is described and exemplified in the Examples presented herein.

The level of LECT2 protein expression may be determined using any methodknown in the art for the measurement of protein levels. Such methodsinclude, for example, electrophoresis, capillary electrophoresis, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions,absorption spectroscopy, a colorimetric assays, spectrophotometricassays, flow cytometry, immunodiffusion (single or double),immunoelectrophoresis, Western blotting, radioimmunoassay (MA),enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays,electrochemiluminescence assays, and the like.

The term “sample” as used herein refers to a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, lymph, urine,cerebrospinal fluid, saliva, ocular fluids, and the like. Tissue samplesmay include samples from tissues, organs or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes). In preferred embodiments, a “sample derived from asubject” refers to blood or plasma drawn from the subject. In furtherembodiments, a “sample derived from a subject” refers to liver tissuederived from the subject.

In some embodiments of the methods of the invention, the antisensepolynucleotide agent is administered to a subject such that theantisense polynucleotide agent is delivered to a specific site withinthe subject. The inhibition of expression of LECT2 may be assessed usingmeasurements of the level or change in the level of LECT2 mRNA or LECT2protein in a sample derived from fluid or tissue from the specific sitewithin the subject. The site may also be a subsection or subgroup ofcells from any one of the aforementioned sites. The site may alsoinclude cells that express a particular type of receptor.

The phrase “contacting a cell with an antisense polynucleotide agent,”as used herein, includes contacting a cell by any possible means.Contacting a cell with an antisense polynucleotide agent includescontacting a cell in vitro with the antisense polynucleotide agent orcontacting a cell in vivo with the antisense polynucleotide agent. Thecontacting may be done directly or indirectly. Thus, for example, theantisense polynucleotide agent may be put into physical contact with thecell by the individual performing the method, or alternatively, theantisense polynucleotide agent may be put into a situation that willpermit or cause it to subsequently come into contact with the cell.

Contacting a cell in vitro may be done, for example, by incubating thecell with the antisense polynucleotide agent. Contacting a cell in vivomay be done, for example, by injecting the antisense polynucleotideagent into or near the tissue where the cell is located, or by injectingthe antisense polynucleotide agent into another area, e.g., thebloodstream or the subcutaneous space, such that the agent willsubsequently reach the tissue where the cell to be contacted is located.For example, the antisense polynucleotide agent may contain and/or becoupled to a ligand, e.g., GalNAc3, that directs the antisensepolynucleotide agent to a site of interest, e.g., the liver.Combinations of in vitro and in vivo methods of contacting are alsopossible. For example, a cell may also be contacted in vitro with anantisense polynucleotide agent and subsequently transplanted into asubject.

In one embodiment, contacting a cell with an antisense polynucleotideagent includes “introducing” or “delivering the antisense polynucleotideagent into the cell” by facilitating or effecting uptake or absorptioninto the cell. Absorption or uptake of an antisense polynucleotide agentcan occur through unaided diffusive or active cellular processes, or byauxiliary agents or devices. Introducing an antisense polynucleotideagent into a cell may be in vitro and/or in vivo. For example, for invivo introduction, antisense polynucleotide agent can be injected into atissue site or administered systemically. In vivo delivery can also bedone by a beta-glucan delivery system, such as those described in U.S.Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No.2005/0281781, the entire contents of which are hereby incorporatedherein by reference. In vitro introduction into a cell includes methodsknown in the art such as electroporation and lipofection. Furtherapproaches are described herein below and/or are known in the art.

VIII. Methods for Treating or Preventing a LECT2-Associated Disorder

The present invention also provides therapeutic and prophylactic methodswhich include administering to a subject having a LECT2-associateddisease, e.g., amyloidosis, e.g. a LECT2 amyloidosis (ALECT2), anantisense polynucleotide agent or pharmaceutical compositions comprisingan antisense polynucleotide agent of the invention. In some aspects ofthe invention, the methods further include administering to the subjectan additional therapeutic agent. In one aspect, the present inventionprovides methods of treating a subject having a disorder that wouldbenefit from reduction in LECT2 expression, e.g., at LECT2-associateddisease, e.g., amyloidosis, e.g. a LECT2 amyloidosis (ALECT2). Thetreatment methods (and uses) of the invention include administering tothe subject, e.g., a human, a therapeutically effective amount of anantisense polynucleotide agent targeting a LECT2 gene or apharmaceutical composition comprising an antisense polynucleotide agenttargeting a LECT2 gene, thereby treating the subject having a disorderthat would benefit from reduction in LECT2 expression.

In one aspect, the invention provides methods of preventing at least onesymptom in a subject having a disorder that would benefit from reductionin LECT2 expression, e.g., a LECT2-associated disease, e.g.,amyloidosis, e.g., a LECT2 amyloidosis (ALECT2). The methods includeadministering to the subject a prophylactically effective amount of anantisense polynucleotide agent targeting a LECT2 gene or apharmaceutical composition comprising an antisense polynucleotide agenttargeting a LECT2 gene, thereby preventing at least one symptom in thesubject having a disorder that would benefit from reduction in LECT2expression.

In yet another aspect, the present invention provides use of anantisense polynucleotide agent of the invention targeting a LECT2 geneor a pharmaceutical composition comprising an antisense polynucleotideagent targeting a LECT2 gene in the manufacture of a medicament fortreating a subject, e.g., a subject that would benefit from a reductionand/or inhibition of LECT2 expression, such as a subject having adisorder that would benefit from reduction in LECT2 expression, e.g., aLECT2-associated disease, e.g., amyloidosis, e.g. a LECT2 amyloidosis(ALECT2).

In another aspect, the invention provides uses of an antisensepolynucleotide agent of the invention for preventing at least onesymptom in a subject suffering from a disorder that would benefit from areduction and/or inhibition of LECT2 expression, such as aLECT2-associated disease, e.g., amyloidosis, e.g. a LECT2 amyloidosis(ALECT2).

In a further aspect, the present invention provides uses of an antisensepolynucleotide agent of the invention in the manufacture of a medicamentfor preventing at least one symptom in a subject suffering from adisorder that would benefit from a reduction and/or inhibition of LECT2expression, such as a LECT2-associated disease, e.g., amyloidosis, e.g.a LECT2 amyloidosis (ALECT2).

“Therapeutically effective amount,” as used herein, is intended toinclude the amount of an antisense polynucleotide agent, that, whenadministered to a subject having a LECT2-associated disease, issufficient to effect treatment of the disease (e.g., by diminishing,ameliorating or maintaining the existing disease or one or more symptomsof disease). The “therapeutically effective amount” may vary dependingon the antisense polynucleotide agent or additional therapeutic agent,how the agent is administered, the disease and its severity and thehistory, age, weight, family history, genetic makeup, the types ofpreceding or concomitant treatments, if any, and other individualcharacteristics of the subject to be treated. Therapeutic effects ofadministration of a LECT2 antsense polynucleotide can be established,for example, by comparison with an appropriate control. For example,inhibition of amyloid deposition may be established, for example, in agroup of patients with amyloidosis (e.g., LECT2 amyloidosis) bycomparison of any appropriate parameter (e.g., a parameter assessing thesize, number, or extent of amyloid deposition) with the same parameterin an appropriate control group. A control group (e.g., a group ofsimilar individuals or the same group of individuals in a crossoverdesign) may include, for example, an untreated population; a populationthat has been treated with a conventional treatment; a population thathas been treated with placebo or a non-targeting iRNA; and the like.

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of an antisense polynucleotide agent that, whenadministered to a subject having a LECT2-associated disease but not yet(or currently) experiencing or displaying symptoms of the disease,and/or a subject at risk of developing a LECT2-associated disease, issufficient to effect treatment of the disease (e.g., by diminishing,ameliorating or maintaining the existing disease or one or more symptomsof disease). Ameliorating the disease includes slowing the course of thedisease or reducing the severity of later-developing disease. The“prophylactically effective amount” may vary depending on the antisensepolynucleotide agent or anti-LECT2 antibody, or antigen-binding fragmentthereof, how the agent or anti-LECT2 antibody, or antigen-bindingfragment thereof, is administered, the degree of risk of disease, andthe history, age, weight, family history, genetic makeup, the types ofpreceding or concomitant treatments, if any, and other individualcharacteristics of the patient to be treated.

A “therapeutically effective amount” or “prophylactically effectiveamount” also includes an amount of an antisense polynucleotide agent,that produces some desired local or systemic effect at a reasonablebenefit/risk ratio applicable to any treatment. Antisense polynucleotideagents employed in the methods of the present invention may beadministered in a sufficient amount to produce a reasonable benefit/riskratio applicable to such treatment.

In another aspect, the present invention provides uses of atherapeutically effective amount of an antisense polynucleotide agent ofthe invention for treating a subject, e.g., a subject that would benefitfrom a reduction and/or inhibition of LECT2 expression.

As used herein, “LECT2 associated disease,” a “disorder that wouldbenefit from reduction in LECT2 expression”, a “pathological processrelated to LECT2 expression,” or the like includes any condition,disorder, or disease in which LECT2 expression is altered (e.g.,decreased or increased relative to a normal level). In some embodiments,LECT2 expression is decreased. In some embodiments, LECT2 expression isincreased. In embodiments, the decrease or increase in LECT2 expressionis detectable in the blood (e.g., in the plasma) of the subject. Inembodiments, the decrease or increase in LECT2 expression is detectablein a tissue sample from the subject (e.g., in a kidney sample or a liversample). The decrease or increase may be assessed relative the levelobserved in the same individual prior to the development of the disorderor relative to other individual(s) who do not have the disorder. Thedecrease or increase may be limited to a particular organ, tissue, orregion of the body (e.g., the kidney or the liver).

In some embodiments, the subject is an animal that serves as a model fora disorder related to LECT2 expression, e.g., a LECT2 amyloidosis. Themethods and uses of the invention include administering a compositiondescribed herein such that expression of the target LECT2 gene isdecreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24, 28,32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or about 80 hours. Inone embodiment, expression of the target LECT2 gene is decreased for anextended duration, e.g., at least about two, three, four, five, six,seven days or more, e.g., about one week, two weeks, three weeks, orabout four weeks or longer.

Administration of the antisense polynucleotide agent according to themethods and uses of the invention may result in a reduction of theseverity, signs, symptoms, and/or markers of such diseases or disordersin a patient with a LECT2-associated disease. By “reduction” in thiscontext is meant a statistically significant decrease in such level. Thereduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, orabout 100%.

Efficacy of treatment or prevention of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, reduction in pain, quality of life, dose of a medicationrequired to sustain a treatment effect, level of a disease marker or anyother measurable parameter appropriate for a given disease being treatedor targeted for prevention. It is well within the ability of one skilledin the art to monitor efficacy of treatment or prevention by measuringany one of such parameters, or any combination of parameters.Comparisons of the later readings with the initial readings provide aphysician an indication of whether the treatment is effective. It iswell within the ability of one skilled in the art to monitor efficacy oftreatment or prevention by measuring any one of such parameters, or anycombination of parameters. In connection with the administration of anantisense polynucleotide agent targeting LECT2 or pharmaceuticalcomposition thereof, “effective against” a LECT2-associated diseaseindicates that administration in a clinically appropriate manner resultsin a beneficial effect for at least a statistically significant fractionof patients, such as improvement of symptoms, a cure, a reduction indisease, extension of life, improvement in quality of life, or othereffect generally recognized as positive by medical doctors familiar withtreating a LECT2-associated disease and the related causes.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given antisense polynucleotide agent drug orformulation of that drug can also be judged using an experimental animalmodel for the given disease as known in the art. When using anexperimental animal model, efficacy of treatment is evidenced when astatistically significant reduction in a marker or symptom is observed.

Alternatively, the efficacy can be measured by a reduction in theseverity of disease as determined by one skilled in the art of diagnosisbased on a clinically accepted disease severity grading scale. Anypositive change resulting in e.g., lessening of severity of diseasemeasured using the appropriate scale, represents adequate treatmentusing an antisense polynucleotide agent or antisense polynucleotideagent formulation as described herein.

Subjects can be administered a therapeutic amount of antisensepolynucleotide agent, such as about 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg,0.04 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg,0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg, 0.45 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.6mg/kg, 0.65 mg/kg, 0.7 mg/kg, 0.75 mg/kg, 0.8 mg/kg, 0.85 mg/kg, 0.9mg/kg, 0.95 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg,2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7mg/kg, 2.8 mg/kg, 2.9 mg/kg, 3.0 mg/kg, 3.1 mg/kg, 3.2 mg/kg, 3.3 mg/kg,3.4 mg/kg, 3.5 mg/kg, 3.6 mg/kg, 3.7 mg/kg, 3.8 mg/kg, 3.9 mg/kg, 4.0mg/kg, 4.1 mg/kg, 4.2 mg/kg, 4.3 mg/kg, 4.4 mg/kg, 4.5 mg/kg, 4.6 mg/kg,4.7 mg/kg, 4.8 mg/kg, 4.9 mg/kg, 5.0 mg/kg, 5.1 mg/kg, 5.2 mg/kg, 5.3mg/kg, 5.4 mg/kg, 5.5 mg/kg, 5.6 mg/kg, 5.7 mg/kg, 5.8 mg/kg, 5.9 mg/kg,6.0 mg/kg, 6.1 mg/kg, 6.2 mg/kg, 6.3 mg/kg, 6.4 mg/kg, 6.5 mg/kg, 6.6mg/kg, 6.7 mg/kg, 6.8 mg/kg, 6.9 mg/kg, 7.0 mg/kg, 7.1 mg/kg, 7.2 mg/kg,7.3 mg/kg, 7.4 mg/kg, 7.5 mg/kg, 7.6 mg/kg, 7.7 mg/kg, 7.8 mg/kg, 7.9mg/kg, 8.0 mg/kg, 8.1 mg/kg, 8.2 mg/kg, 8.3 mg/kg, 8.4 mg/kg, 8.5 mg/kg,8.6 mg/kg, 8.7 mg/kg, 8.8 mg/kg, 8.9 mg/kg, 9.0 mg/kg, 9.1 mg/kg, 9.2mg/kg, 9.3 mg/kg, 9.4 mg/kg, 9.5 mg/kg, 9.6 mg/kg, 9.7 mg/kg, 9.8 mg/kg,9.9 mg/kg, 9.0 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg,35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg ,70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, or about 100mg/kg. Values and ranges intermediate to the recited values are alsointended to be part of this invention.

In certain embodiments, for example, when a composition of the inventioncomprises a antisense polynucleotide agent as described herein and alipid, subjects can be administered a therapeutic amount of antisensepolynucleotide agent, such as about 0.01 mg/kg to about 5 mg/kg, about0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg to about 5 mg/kg, about0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3mg/kg to about 10 mg/kg, about 0.4 mg/kg to about 5 mg/kg, about 0.4mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kgto about 10 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 1.5 mg/kg toabout 10 mg/kg, about 2 mg/kg to about 2.5 mg/kg, about 2 mg/kg to about10 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 10mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 5 mg/kg,about 4.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 10 mg/kg, about4.5 mg/kg to about 10 mg/kg, about 5 mg/kg to about 10 mg/kg, about 5.5mg/kg to about 10 mg/kg, about 6 mg/kg to about 10 mg/kg, about 6.5mg/kg to about 10 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7.5mg/kg to about 10 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5mg/kg to about 10 mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5mg/kg to about 10 mg/kg. Values and ranges intermediate to the recitedvalues are also intended to be part of this invention.

For example, the antisense polynucleotide agent may be administered at adose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10mg/kg. Values and ranges intermediate to the recited values are alsointended to be part of this invention.

In other embodiments, for example, when a composition of the inventioncomprises a antisense polynucleotide agent as described herein and anN-acetylgalactosamine, subjects can be administered a therapeutic amountof antisense polynucleotide agent, such as a dose of about 0.1 to about100 mg/kg, about 0.25 to about 100 mg/kg, about 0.5 to about 100 mg/kg,about 0.75 to about 100 mg/kg, about 1 to about 100 mg/mg, about 1.5 toabout 100 mg/kg, about 2 to about 100 mg/kg, about 2.5 to about 100mg/kg, about 3 to about 100 mg/kg, about 3.5 to about 100 mg/kg, about 4to about 100 mg/kg, about 4.5 to about 100 mg/kg, about 5 to about 100mg/kg, about 7.5 to about 100 mg/kg, about 10 to about 100 mg/kg, about15 to about 100 mg/kg, about 20 to about 100 mg/kg, about 25 to about100 mg/kg, about 30 to about 100 mg/kg, about 35 to about 100 mg/kg,about 40 to about 100 mg/kg, about 45 to about 100 mg/kg, about 0.1 toabout 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45 mg/kg, about0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1 to about 40mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg, about0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20mg/kg, or about 15 to about 20 mg/kg. In one embodiment, when acomposition of the invention comprises a antisense polynucleotide agentas described herein and an N-acetylgalactosamine, subjects can beadministered a therapeutic amount of about 10 to 100 or about 0.5 toabout 10 mg/kg of antisense polynucleotide agent. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

For example, subjects can be administered a therapeutic amount ofantisense polynucleotide agent, such as about 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8,8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5,15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5,22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5,29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 67, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or about 100 mg/kg. Values and ranges intermediate to therecited values are also intended to be part of this invention.

The antisense polynucleotide agent can be administered by intravenousinfusion over a period of time, such as over a 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or about a 25 minuteperiod. The administration may be repeated, for example, on a regularbasis, such as weekly, biweekly (i.e., every two weeks) for one month,two months, three months, four months or longer. After an initialtreatment regimen, the treatments can be administered on a less frequentbasis. For example, after administration weekly or biweekly for threemonths, administration can be repeated once per month, for six months ora year or longer.

Administration of the antisense polynucleotide agent can reduce LECT2levels, e.g., in a cell, tissue, blood, urine or other compartment ofthe patient by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or at least about 99% or more.

Before administration of a full dose of the antisense polynucleotideagent, patients can be administered a smaller dose, such as a 5%infusion, and monitored for adverse effects, such as an allergicreaction. In another example, the patient can be monitored for unwantedimmunostimulatory effects, such as increased cytokine (e.g., TNF-alphaor INF-alpha) levels.

Owing to the inhibitory effects on LECT2 expression, a compositionaccording to the invention or a pharmaceutical composition preparedtherefrom can enhance the quality of life.

An antisense polynucleotide agent of the invention may be administeredin “naked” form, or as a “free antisense polynucleotide agent.” A nakedantisense polynucleotide agent is administered in the absence of apharmaceutical composition. The naked antisense polynucleotide agent maybe in a suitable buffer solution. The buffer solution may compriseacetate, citrate, prolamine, carbonate, or phosphate, or any combinationthereof. In one embodiment, the buffer solution is phosphate bufferedsaline (PBS). The pH and osmolarity of the buffer solution containingthe antisense polynucleotide agent can be adjusted such that it issuitable for administering to a subject.

Alternatively, an antisense polynucleotide agent of the invention may beadministered as a pharmaceutical composition, such as an antisensepolynucleotide agent liposomal formulation.

The invention further provides methods and uses of an antisensepolynucleotide agent or a pharmaceutical composition thereof (includingmethods and uses of an antisense polynucleotide agent or apharmaceutical composition comprising an antisense polynucleotide agentand an anti-LECT2 antibody, or antigen-bidning fragment thereof) fortreating a subject that would benefit from reduction and/or inhibitionof LECT2 expression, e.g., a subject having a LECT2-associated disease,in combination with other pharmaceuticals and/or other therapeuticmethods, e.g., with known pharmaceuticals and/or known therapeuticmethods, such as, for example, those which are currently employed fortreating these disorders. For example, in certain embodiments, anantisense polynucleotide agent targeting LECT2 is administered incombination with, e.g., an agent useful in treating a LECT2-associateddisease as described elsewhere herein.

The antisense polynucleotide agent (and/or an anti-LECT2 antibody) andan additional therapeutic agent and/or treatment may be administered atthe same time and/or in the same combination, e.g., parenterally, or theadditional therapeutic agent can be administered as part of a separatecomposition or at separate times and/or by another method known in theart or described herein.

The present invention also provides methods of using an antisensepolynucleotide agent of the invention and/or a composition containing anantisense polynucleotide agent of the invention to reduce and/or inhibitLECT2 expression in a cell. In other aspects, the present inventionprovides an antisense polynucleotide agent of the invention and/or acomposition comprising an antisense polynucleotide agent of theinvention for use in reducing and/or inhibiting LECT2 expression in acell. In yet other aspects, use of an antisense polynucleotide agent ofthe invention and/or a composition comprising an antisensepolynucleotide agent of the invention for the manufacture of amedicament for reducing and/or inhibiting LECT2 expression in a cell areprovided.

The methods and uses include contacting the cell with an antisensepolynucleotide agent, e.g., a antisense polynucleotide agent, of theinvention and maintaining the cell for a time sufficient to obtainantisense inhibition of a LECT2 gene, thereby inhibiting expression ofthe LECT2 gene in the cell.

Reduction in gene expression can be assessed by any methods known in theart. For example, a reduction in the expression of LECT2 may bedetermined by determining the mRNA expression level of LECT2 usingmethods routine to one of ordinary skill in the art, e.g., Northernblotting, qRT-PCR, by determining the protein level of LECT2 usingmethods routine to one of ordinary skill in the art, such as Westernblotting, immunological techniques, flow cytometry methods, and/orELISA.

In the methods and uses of the invention the cell may be contacted invitro or in vivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may beany cell that expresses a LECT2 gene. A cell suitable for use in themethods and uses of the invention may be a mammalian cell, e.g., aprimate cell (such as a human cell or a non-human primate cell, e.g., amonkey cell or a chimpanzee cell), a non-primate cell (such as a cowcell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell,a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, adog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bearcell, or a buffalo cell), a bird cell (e.g., a duck cell or a goosecell), or a whale cell. In one embodiment, the cell is a human cell,e.g., a human liver cell.

LECT2 expression may be inhibited in the cell by at least about 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%.

The in vivo methods and uses of the invention may include administeringto a subject a composition containing an antisense polynucleotide agent,where the antisense polynucleotide agent includes a nucleotide sequencethat is complementary to at least a part of an RNA transcript of theLECT2 gene of the mammal to be treated. When the organism to be treatedis a mammal such as a human, the composition can be administered by anymeans known in the art including, but not limited to subcutaneous,intravenous, oral, intraperitoneal, or parenteral routes, includingintracranial (e.g., intraventricular, intraparenchymal and intrathecal),intramuscular, transdermal, airway (aerosol), nasal, rectal, and topical(including buccal and sublingual) administration. In certainembodiments, the compositions are administered by subcutaneous orintravenous infusion or injection.

In some embodiments, the administration is via a depot injection. Adepot injection may release the antisense polynucleotide agent in aconsistent way over a prolonged time period. Thus, a depot injection mayreduce the frequency of dosing needed to obtain a desired effect, e.g.,a desired inhibition of LECT2, or a therapeutic or prophylactic effect.A depot injection may also provide more consistent serum concentrations.Depot injections may include subcutaneous injections or intramuscularinjections. In preferred embodiments, the depot injection is asubcutaneous injection.

In some embodiments, the administration is via a pump. The pump may bean external pump or a surgically implanted pump. In certain embodiments,the pump is a subcutaneously implanted osmotic pump. In otherembodiments, the pump is an infusion pump. An infusion pump may be usedfor intravenous, subcutaneous, arterial, or epidural infusions. Inpreferred embodiments, the infusion pump is a subcutaneous infusionpump. In other embodiments, the pump is a surgically implanted pump thatdelivers the antisense polynucleotide agent to the liver.

The mode of administration may be chosen based upon whether local orsystemic treatment is desired and based upon the area to be treated. Theroute and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods forinhibiting the expression of a LECT2 gene in a mammal, e.g., a human.The present invention also provides a composition comprising anantisense polynucleotide agent that targets a LECT2 gene in a cell of amammal for use in inhibiting expression of the LECT2 gene in the mammal.In another aspect, the present invention provides use of an antisensepolynucleotide agent that targets a LECT2 gene in a cell of a mammal inthe manufacture of a medicament for inhibiting expression of the LECT2gene in the mammal.

The methods and uses include administering to the mammal, e.g., a human,a composition comprising an antisense polynucleotide agent that targetsa LECT2 gene in a cell of the mammal and maintaining the mammal for atime sufficient to obtain antisense inhibition of the mRNA transcript ofthe LECT2 gene, thereby inhibiting expression of the LECT2 gene in themammal.

Reduction in gene expression can be assessed by any methods known it theart and by methods, e.g. qRT-PCR, described herein. Reduction in proteinproduction can be assessed by any methods known it the art and bymethods, e.g., ELISA or Western blotting, described herein.

In one embodiment, a puncture liver biopsy sample serves as the tissuematerial for monitoring the reduction in LECT2 gene and/or proteinexpression. In another embodiment, a blood sample serves as the tissuematerial for monitoring the reduction in LECT2 gene and/or proteinexpression. Suitable assays are further described in the Examplessection below.

A. LECT2 Amyloidosis

In embodiments, the disorder related to LECT2 expression is anamyloidosis, e.g., a LECT2 amyloidosis. LECT2 amyloidosis has beendescribed in several clinical studies. See, e.g., Benson, M. D. et al(2008) Kidney International, 74: 218-222; Murphy, C. L. et al. (2010) AmJ Kidney Dis, 56(6):1100-1107; Larsen, C. P. et al. (2010) Kidney Int.,77(9):816-819; Holanda, D. G. et al. (20011) Nephrol. Dial. Transplant.,26 (1): 373-376; and Sethi, S. et al. (2012) Kidney International 82,226-234 (hereinafter Sethi et al.).

Clinical and pathological features of LECT2 amyloidosis mimic those ofamyloid light chain (AL) amyloidosis. These symptoms include, e.g.,symptoms of kidney disease and renal failure, e.g., fluid retention,swelling, and shortness of breath. Amyloidosis may affect the heart,peripheral nervous system, gastrointestinal tract, blood, lungs andskin. Heart complications include, e.g., heart failure and irregularheart beat. Other symptoms include, e.g., stroke, gastrointestinaldisorders, enlarged liver, diminished spleen function, diminishedfunction of the adrenal and other endocrine glands, skin color change orgrowths, lung problems, bleeding and bruising problems, fatigue andweight loss. In embodiments, the methods described herein are associatedwith improvement in one or more symptoms described herein.

Methods for diagnosis of amyloidosis, e.g., LECT2 amyloidosis, aredescribed, e.g., in Leung, N. et al. (2010) Blood, published online Sep.4, 2012; DOI 10.1182/blood-2012-03-413682; Shiller, S. M. et al. (2011).Laboratory Methods for the Diagnosis of Hereditary Amyloidoses,Amyloidosis—Mechanisms and Prospects for Therapy, Dr. SvetlanaSarantseva (Ed.), ISBN: 978-953-307-253-1; Sethi et al. (see above) andin U.S. Patent Application Publication No. 20100323381.

Based on the results provided by Sethi et al., LECT2 amyloidosisaccounts for a significant percentage of cases of renal amyloidosis. SeeTable 1 of Sethi et al., which shows that 26 out of 127 cases of renalamyloidosis studied by laser microdissection and mass spectrometry ofrenal biopsy and/or nephrectomy specimens were determined to be of theLECT2 amyloid type. Sethi et al. further report that apolipoprotein Eprotein and serum amyloid P component (SAP) were also present in allcases of LECT2 amyloidosis.

In embodiments, the amyloidosis, e.g., the LECT2 amyloidosis, involvessystemic amyloid deposition. In embodiments, the amyloidosis, e.g., theLECT2 amyloidosis, is localized entirely or predominately to aparticular tissue or organ (e.g., to the kidney or liver).

In embodiments, the amyloidosis, e.g., the LECT2 amyloidosis, ishereditary.

In embodiments, a LECT2 amyoidosis is diagnosed using analysis of asample from the subject (e.g., a biopsy sample). In embodiments, thebiopsy sample is a renal biopsy. In embodiments, the sample is anephrectomy sample. In embodiments, the sample is from a liver biopsy orfrom other resected liver tissue. In embodiments, the sample is analyzedusing methods selected from one or more of immunohistochemistry, LECT2immunoassay, electron microscopy, laser microdissection, and massspectrometry. In embodiments, the LECT2 amyloidosis is diagnosed usinglaser microdissection and mass spectrometry.

In embodiments, the amyloidosis, e.g., the LECT2 amyloidosis, affectsthe kidney, e.g., involves amyloid deposition in the kidney. Inembodiments, kidney function is compromised as a result of theamyloidosis. In embodiments, the subject suffers from one or more offluid retention, swelling, and shortness of breath. In embodiments, thesubject has nephrotic syndrome. In embodiments, the subject suffers fromproteinuria. In embodiments, the subject has renal failure.

In embodiments, the amyloidosis, e.g., the LECT2 amyloidosis, affectsthe liver, e.g., involves amyloid deposition in the liver. Inembodiments, liver function is compromised as a result of theamyloidosis. In embodiments, the subject has hepatitis, e.g., chronichepatitis. In embodiments, the hepatitis is a viral hepatitis.

LECT2 amyloidosis has been found to be particularly prevalent in MexicanAmericans and has also been associated with homozygosity for the Gallele of the LECT2 gene that encodes valine at position 40 in themature protein (amino acid 58 in the unprocessed protein). See, e.g.,Benson, M. D. et al. (2008) Kidney International, 74: 218-222; Murphy,C. L. et al. (2010) Am J Kidney Dis, 56(6):1100-1107.

In some embodiments, the subject is of Mexican descent. In someembodiments, the subject is a Mexican American.

In embodiments, the subject carries the G allele of the LECT2 gene thatencodes valine at position 40 in the mature protein (amino acid 58 inthe unprocessed protein). In embodiments, the subject is homozygous forthe G allele (G/G genotype). In embodiments, a LECT2 protein expressedin the subject has valine at position 40 in the mature protein (or atamino acid 58 in the unprocessed protein).

In some embodiments, the method decreases LECT2 expression. Inembodiments, the decrease in LECT2 expression is assessed relative tothe level in the same individual prior to the treatment. In someembodiments, the method is shown to decrease LECT2 expression bycomparing the levels of LECT2 expression in a treated subject (or groupof subjects) with the levels in a control subject (or group ofsubjects), e.g., an untreated subject (or group of subjects) or asubject (or group of subjects) treated with a control treatment (e.g.,an iRNA (e.g., a dsRNA) that does not target LECT2).

In embodiments, the method reduces amyloid deposition, e.g., depositionof amyloid comprising a LECT2 protein or a portion thereof. Inembodiments, the protein is a wild type protein. In embodiments, theprotein is a human LECT2 protein, or a portion thereof, that includesvaline at position 40 (position 40 of the mature, secreted protein, orat amino acid 58 in the unprocessed protein, as described herein). Inembodiments, the method decreases the size, number, and/or extent ofamyloid deposits.

In embodiments, the method decreases one or more symptoms associatedwith amyloid deposition.

In some embodiments, the antisense polynucleotide is administered in aform that targets the dsRNA to a particular organ or tissue to inhibitamyloid deposition in the organ or tissue.

In some embodiments, the antisense polynucleotide is targeted to theliver. In some embodiments, the dsRNA is conjugated to a ligand, e.g., aGalNAc ligand (e.g., a GalNAc ligand as described herein) that targetsthe dsRNA to the liver (e.g., to hepatocytes).

Also provided herein is a method of reducing amyloid deposition, themethod comprising administering antisense polynucleotide agent asdisclosed herein to a subject in need thereof (e.g., a subject having,suspected of having, or at risk for developing a LECT2 amyloidosis). Inembodiments, the method decreases (e.g., prevents or diminishes) thesize, number, and/or extent of amyloid deposits. The size, number,and/or extent of amyloid deposits may be assessed using any method knownin the art (e.g., immunoassay, immunohistochemistry, mass spectrometry).The reduction of amyloid deposition may involve a decrease in amyloiddeposition (e.g., size, number, and/or extent of amyloid deposits) of atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more.

In the methods provided herein, the antisense polynucleotide andcompositions thereof are administered in a therapeutically effectiveamount.

B. Rheumatoid Arthritis

Rheumatoid arthritis is also a disorder related to LECT2 expression. Inparticular, in a Japanese population, it was found that possession ofone A allele of the LECT2 gene that encodes isoleucine at position 40 inthe mature protein (or amino acid 58 in the unprocessed protein) wasfound to increase the overall risk of developing rheumatoid arthritis.Possessing two A alleles was strongly associated with disease severity.See Kameoka, Y. et al. (2000) Arth Rheum, 43 (6): 1419-20.

In one embodiment of the methods provided herein, the disorder relatedto LECT2 expression is rheumatoid arthritis. In one embodiment, theantisense polynucleotide agent inhibits LECT2 expression in a subjecthaving rheumatoid arthritis. In some such embodiments, the antisensepolynucleotide agent inhibits LECT2 expression in synovial tissue and/orin synovial fluid-derived cells (e.g., mononuclear cells andfibroblasts). In some embodiments, the antisense polynucleotide agenttargets an mRNA that encodes isoleucine at position 40 in the matureprotein (amino acid 58 in the unprocessed protein).

C. Liver Injury

LECT2 expression can increase during acute liver injury.

In one embodiment of the methods provided herein, the disorder relatedto LECT2 expression is acute liver injury. In embodiments, the antisensepolynucleotide agent modulates (e.g., increases or decreases) LECT2expression. In embodiments, the antisense polynucleotide agent modulatesLECT2 expression in the liver. In embodiments, the antisensepolynucleotide agent decreases LECT2 expression in the liver. Inembodiments, the antisense polynucleotide agent increases LECT2expression in the liver.

D. Combination Therapies

In another aspect, the present invention provides uses of atherapeutically effective amount of an antisense polynucleotide agent ofthe invention and an additional therapeutic agent, for treating asubject, e.g., a subject that would benefit from a reduction and/orinhibition of LECT2 expression.

In embodiments, an antisense polynucleotide agent (e.g., a dsRNA)disclosed herein is administered in combination with an additionaltherapeutic agent (e.g., one or more additional therapies) known to beeffective in treating a disorder related to LECT2 expression (e.g., aLECT2 amyloidosis) or a symptom of such a disorder. The antisensepolynucleotide agent may be administered before, after, or concurrentwith the additional therapeutic agent. In embodiments, the antisensepolynucleotide agent is administered before the additional therapeuticagent. In embodiments, the antisense polynucleotide agent isadministered after the additional therapeutic agent. In embodiments, theantisense polynucleotide agent is administered concurrent with theadditional therapeutic agent.

In another aspect, the invention provides methods of preventing at leastone symptom in a subject having a disorder that would benefit fromreduction in LECT2 expression, e.g., a LECT2-associated disease, e.g.,amyloidosis, e.g. a LECT2 amyloidosis (ALECT2). The methods includeadministering to the subject a prophylactically effective amount of anantisense polynucleotide agent targeting a LECT2 gene or apharmaceutical composition comprising an antisense polynucleotide agenttargeting a LECT2 gene, and an additional therapeutic agent, therebypreventing at least one symptom in the subject having a disorder thatwould benefit from reduction in LECT2 expression.

In another aspect, the present invention provides methods of treating asubject having a disorder that would benefit from reduction in LECT2expression, e.g., a LECT2-associated disease, e.g., amyloidosis, e.g. aLECT2 amyloidosis (ALECT2), which include administering to the subject,e.g., a human, a therapeutically effective amount of an antisensepolynucleotide agent targeting a LECT2 gene or a pharmaceuticalcomposition comprising an antisense polynucleotide agent targeting aLECT2 gene, and an additional therapeutic agent, thereby treating thesubject having a disorder that would benefit from reduction in LECT2expression.

In another aspect, the present invention provides uses of an antisensepolynucleotide agent of the invention targeting a LECT2 gene or apharmaceutical composition comprising an antisense polynucleotide agenttargeting a LECT2 gene in the manufacture of a medicament for use incombination with an additional therapeutic agent, for treating asubject, e.g., a subject that would benefit from a reduction and/orinhibition of LECT2 expression, e.g., a LECT2-associated disease, e.g.,amyloidosis, e.g. a LECT2 amyloidosis (ALECT2).

In yet another aspect, the invention provides uses of an antisensepolynucleotide agent of the invention, and an additional therapeuticagent, for preventing at least one symptom in a subject suffering from adisorder that would benefit from a reduction and/or inhibition of LECT2expression, such as a LECT2-associated disease, e.g., amyloidosis, e.g.a LECT2 amyloidosis (ALECT2).

In a further aspect, the present invention provides uses of an antisensepolynucleotide agent of the invention in the manufacture of a medicamentfor use in combination with an additional therapeutic agent, forpreventing at least one symptom in a subject suffering from a disorderthat would benefit from a reduction and/or inhibition of LECT2expression, such as a LECT2-associated disease, e.g., amyloidosis, e.g.a LECT2 amyloidosis (ALECT2).

In one embodiment, an antisense polynucleotide agent targeting LECT2 isadministered to a subject having a LECT2-associated disease such thatLECT2 levels, e.g., in a cell, tissue, blood, urine or other tissue orfluid of the subject are reduced by at least about 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or at least about 99% or more and, subsequently, an additionaltherapeutic (as described below) is administered to the subject.

In embodiments, the additional therapeutic agent is a second therapy.The agent and the additional therapeutic agent can be administered incombination in the same composition or the additional therapeutic agentcan be administered as part of a separate composition.

In some embodiments, the second therapy is a non-antisensepolynucleotide agent therapeutic agent that is effective to treat thedisorder or symptoms of the disorder.

In some embodiments, the disorder to be treated by the compositions ormethods disclosed herein is a LECT2 amyloidosis that affects kidneyfunction, e.g., through amyloid deposition in the kidney. In some suchembodiments, the agent is administered in conjunction with a therapythat supports kidney function (e.g., dialysis, a diuretic, anangiotensin converting enzyme (ACE) inhibitor, an angiotensin receptorblocker (ARB), or dialysis).

In some embodiments, the disorder to be treated by the compositions ormethods disclosed herein is a LECT2 amyloidosis involving amyloiddeposits in the liver. In some such embodiments, the agent isadministered in conjunction with a therapy that supports liver function.

In some embodiments, the disorder to be treated by the compositions ormethods disclosed herein is a LECT2 amyloidosis, and the agent isadministered in conjunction with removal of all or part of the organ(s)affected by the amyloidosis (e.g., resection of all or part of kidney orliver tissue affected by the amyloidosis). The removal is optionallyconducted in conjunction with a replacement of all or part of the organremoved (e.g., in conjunction with a kidney or liver organ transplant).

In some embodiments, the additional therapeutic agent is an anti-LECT2antibody, or antigen binding fragment thereof.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the antisense polynucleotide agents and methodsfeatured in the invention, suitable methods and materials are describedbelow. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

EXAMPLES Example 1 Antisense Synthesis

The antisense polynucleotides targeting LECT2 were synthesized usingstandard synthesis methods known in the art.

A detailed list of modified and unmodified antisense molecules targetingLECT2 is shown in Table 3, including the starting position and mRNAsequence of the antisense oligonucleotide relative to SEQ ID NO: 1.

Example 2 In vitro Screening

In vitro screening of the antisense polynucleotides was performed bytransfecting human primary hepatocytes with a single 500 nM dose of anantisense LECT2 polynucleotide using methods known in the art. 32000cells/well in a 96 well plate were transfected for 24 hours in 0.4 μlRNAiMAX/well. A single dose transfection screen in cells transfectedwith the indicated antisense polynucleotide is shown in Table 4. Therelative LECT2 mRNA expression relative to a mock transfected controlwas determined.

TABLE 2 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) AAdenosine-3′-phosphate Af 2′-fluoroadenosine-3′-phosphate Afs2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioatea 2′-O-methyladenosine-3′-phosphate as2′-O-methyladenosine-3′-phosphorothioate C cytidine-3′-phosphate dA2′-deoxyadenosine-3′-phosphate dAs 2′-deoxyadenosine-3′-phosphorothioateCf 2′-fluorocytidine-3′-phosphate Cfs2′-fluorocytidine-3′-phosphorothioate Cs cytidine-3′-phosphorothioate c2′-O-methylcytidine-3′-phosphate cs2′-O-methylcytidine-3′-phosphorothioate dC 2′-deoxycytidine-3′-phosphatedCs 2′-deoxycytidine-3′-phosphorothioate G guanosine-3′-phosphate Gf2′-fluoroguanosine-3′-phosphate Gfs2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioateg 2′-O-methylguanosine-3′-phosphate gs2′-O-methylguanosine-3′-phosphorothioate dG2′-deoxyguanosine-3′-phosphate dGs 2′-deoxyguanosine-3′-phosphorothioateT 5′-methyluridine-3′-phosphate f 2′-fluoro-5-methyluridine-3′-phosphateTfs 2′-fluoro-5-methyluridine-3′-phosphorothioate Ts5-methyluridine-3′-phosphorothioate t2′-O-methyl-5-methyluridine-3′-phosphate ts2′-O-methyl-5-methyluridine-3′-phosphorothioate dT2′-deoxythymidine-3′-phosphate dTs 2′-deoxythymidine-3′-phosphorothioateU Uridine-3′-phosphate Uf 2′-fluorouridine-3′-phosphate Ufs2′-fluorouridine-3′-phosphorothioate Us uridine-3′-phosphorothioate u2′-O-methyluridine-3′-phosphate us2′-O-methyluridine-3′-phosphorothioate dU 2′-deoxyuridine-3′-phosphatedUs 2′-deoxyuridine-3′-phosphorothioate S phosphorothioate linkage N anynucleotide (G, A, C, T or U) L96N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinolHyp-(GalNAc-alkyl)3 (dt) deoxy-thymine (5MdC)5′-methyl-deoxycytidine-3′-phosphate (5MdC)s5′-methyl-deoxycytidine-3′-phosphorothioate

TABLE 3 Antisense polynucleotides targeting LECT2. SEQ SEQ SEQ ID ID IDUnmodified Antisense Oligo NO: Modified Antisense Sequence (5′ to 3′)Pos NO: mRNA Target Sequence NO: Sequence A-133875.1  13ususcsusasdAsdTsdAsdTsdTs(5MdC)s(5MdC)sdAsdTsdGsgsasusasg   12 109CUAUCCAUGGAAUAUUAGAA 205 UUCUAAUAUUCCAUGGAUAG A-133876.1  14gsasgscsasdAsdGsdTs(5MdC)sdAsdAsdGsdTsdTs(5MdC)susasasusa   24 110UAUUAGAACUUGACUUGCUC 206 GAGCAAGUCAAGUUCUAAUA A-133877.1  15asasgsasgsdGsdAsdTsdGsdGsdAsdGs(5MdC)sdAsdAsgsuscsasa   33 111UUGACUUGCUCCAUCCUCUU 207 AAGAGGAUGGAGCAAGUCAA A-133878.1  16ascsasasasdAsdAsdGsdTsdTsdTsdAsdAsdGsdAsgsgsasusg   44 112CAUCCUCUUAAACUUUUUGU 208 ACAAAAAGUUUAAGAGGAUG A-133879.1  17usususasgsdTsdGsdTsdGsdAsdGsdAs(5MdC)sdAs(5MdC)sasasasasa   57 113UUUUUGUGUCUCACACUAAA 209 UUUAGUGUGAGACACAAAAA A-133880.1  18uscsuscsasdTsdTsdTs(5MdC)sdTsdTsdTsdAsdGsdTsgsusgsasg   66 114CUCACACUAAAGAAAUGAGA 210 UCUCAUUUCUUUAGUGUGAG A-133881.1  19gsasasusus(5MdC)sdTsdGs(5MdC)sdAsdTs(5MdC)sdTs(5MdC)sdTscsasususu   78115 AAAUGAGAGAUGCAGAAUUC 211 GAAUUCUGCAUCUCUCAUUU A-133882.1  20usasusususdAsdGs(5MdC)s(5MdC)sdTsdTsdAsdGsdAsdAsususcsusg   90 116CAGAAUUCUAAGGCUAAAUA 212 UAUUUAGCCUUAGAAUUCUG A-133883.1  21ascsususcs(5MdC)sdTsdAsdGs(5MdC)sdTsdAsdTsdTsdTsasgscscsu  100 117AGGCUAAAUAGCUAGGAAGU 213 ACUUCCUAGCUAUUUAGCCU A-133884.1  22gsusususgsdAsdAsdTsdGsdAsdAsdTsdAs(5MdC)sdTsuscscsusa  112 118UAGGAAGUAUUCAUUCAAAC 214 GUUUGAAUGAAUACUUCCUA A-133885.1  23asasusasusdTs(5MdC)sdAsdAsdGsdTsdTsdTsdGsdAsasusgsasa  121 119UUCAUUCAAACUUGAAUAUU 215 AAUAUUCAAGUUUGAAUGAA A-133886.1  24uscsuscsusdTsdTsdGsdAsdAsdGsdAsdAsdTsdAsususcsasa  132 120UUGAAUAUUCUUCAAAGAGA 216 UCUCUUUGAAGAAUAUUCAA A-133887.1  25gsususgscs(5MdC)s(5MdC)s(5MdC)s(5MdC)sdAs(5MdC)sdAs(5MdC)sdTs(5MdC)  145121 AAAGAGAGUGUGGGGGCAAC 217 GUUGCCCCCACACUCUCUUU suscsususu A-133888.1 26 csusgsasusdTsdAsdGsdAsdGsdTsdTsdGs(5MdC)s(5MdC)scscscsasc  154 122GUGGGGGCAACUCUAAUCAG 218 CUGAUUAGAGUUGCCCCCAC A-133889.1  27usasgsususdTs(5MdC)sdTsdTs(5MdC)s(5MdC)sdTs(5MdC)sdTsdGsasususasg  166123 CUAAUCAGAGGAAGAAACUA 219 UAGUUUCUUCCUCUGAUUAG A-133890.1  28usususascsdTsdTs(5MdC)s(5MdC)sdTsdTsdTsdAsdGsdTsususcsusu  177 124AAGAAACUAAAGGAAGUAAA 220 UUUACUUCCUUUAGUUUCUU A-133891.1  29asascsasus(5MdC)sdTsdGsdGsdTsdTsdTsdTsdAs(5MdC)sususcscsu  187 125AGGAAGUAAAACCAGAUGUU 221 AACAUCUGGUUUUACUUCCU A-133892.1  30gsgscsususdTsdGsdGsdTsdGsdGsdAsdAsdAsdAscsasuscsu  200 126AGAUGUUUUCCACCAAAGCC 222 GGCUUUGGUGGAAAACAUCU A-133893.1  31gscscsasasdAsdAsdGsdGsdAsdGsdGsdGs(5MdC)sdTsususgsgsu  211 127ACCAAAGCCCUCCUUUUGGC 223 GCCAAAAGGAGGGCUUUGGU A-133894.1  32asasuscsasdGsdAs(5MdC)s(5MdC)sdAsdGs(5MdC)s(5MdC)sdAsdAsasasgsgsa  221128 UCCUUUUGGCUGGUCUGAUU 224 AAUCAGACCAGCCAAAAGGA A-133895.1  33gsusgscsgsdGsdTsdAsdGsdAsdAsdAsdTs(5MdC)sdAsgsascscsa  231 129UGGUCUGAUUUCUACCGCAC 225 GUGCGGUAGAAAUCAGACCA A-133896.1  34asusgsgscs(5MdC)s(5MdC)sdTsdGs(5MdC)s(5MdC)sdAsdGsdTsdGscsgsgsusa  243130 UACCGCACUGGCAGGGCCAU 226 AUGGCCCUGCCAGUGCGGUA A-133897.1  35usasusasusdTsdAsdGs(5MdC)s(5MdC)s(5MdC)sdAsdTsdGsdGscscscsusg  254 131CAGGGCCAUGGGCUAAUAUA 227 UAUAUUAGCCCAUGGCCCUG A-133898.1  36csususgscs(5MdC)sdAsdGs(5MdC)sdAs(5MdC)sdAsdTsdAsdTsasususasg  266 132CUAAUAUAUGUGCUGGCAAG 228 CUUGCCAGCACAUAUAUUAG A-133899.1  37csasususgsdGsdAsdAsdGsdAs(5MdC)sdTsdTsdGs(5MdC)scsasgscsa  276 133UGCUGGCAAGUCUUCCAAUG 229 CAUUGGAAGACUUGCCAGCA A-133900.1  38ascsgsuscs(5MdC)sdGsdGsdAsdTs(5MdC)sdTs(5MdC)sdAsdTsusgsgsasa  288 134UUCCAAUGAGAUCCGGACGU 230 ACGUCCGGAUCUCAUUGGAA A-133901.1  39asusgsgscsdGsdGsdTs(5MdC)sdAs(5MdC)sdAs(5MdC)sdGsdTscscsgsgsa  299 135UCCGGACGUGUGACCGCCAU 231 AUGGCGGUCACACGUCCGGA A-133902.1  40gsuscscsas(5MdC)sdAsdGs(5MdC)s(5MdC)sdAsdTsdGsdGs(5MdC)sgsgsuscsa  309136 UGACCGCCAUGGCUGUGGAC 232 GUCCACAGCCAUGGCGGUCA A-133903.1  41gsasgscsasdGsdAsdGsdTsdAs(5MdC)sdTsdGsdTs(5MdC)scsascsasg  321 137CUGUGGACAGUACUCUGCUC 233 GAGCAGAGUACUGUCCACAG A-133904.1  42usgsascsusdTs(5MdC)sdTsdTsdTsdGsdAsdGs(5MdC)sdAsgsasgsusa  331 138UACUCUGCUCAAAGAAGUCA 234 UGACUUCUUUGAGCAGAGUA A-133905.1  43gsusgsasgsdGs(5MdC)s(5MdC)sdTs(5MdC)sdTsdGsdAs(5MdC)sdTsuscsususu  341139 AAAGAAGUCAGAGGCCUCAC 235 GUGAGGCCUCUGACUUCUUU A-133906.1  44uscscsascsdAs(5MdC)s(5MdC)s(5MdC)sdTsdGsdGsdTsdGsdAsgsgscscsu  352 140AGGCCUCACCAGGGUGUGGA 236 UCCACACCCUGGUGAGGCCU A-133907.1  45gsasgscsas(5MdC)sdAsdAsdGsdAsdTsdGsdTs(5MdC)s(5MdC)sascsascsc  364 141GGUGUGGACAUCUUGUGCUC 237 GAGCACAAGAUGUCCACACC A-133908.1  46asgsasuscs(5MdC)sdAsdGs(5MdC)sdAsdGsdAsdGs(5MdC)sdAscsasasgsa  374 142UCUUGUGCUCUGCUGGAUCU 238 AGAUCCAGCAGAGCACAAGA A-133909.1  47usgscsgsusdAs(5MdC)sdAs(5MdC)sdAsdGsdTsdAsdGsdAsuscscsasg  386 143CUGGAUCUACUGUGUACGCA 239 UGCGUACACAGUAGAUCCAG A-133910.1  48csasgsusgsdAsdAsdTsdGsdGsdTsdGs(5MdC)sdGsdTsascsascsa  396 144UGUGUACGCACCAUUCACUG 240 CAGUGAAUGGUGCGUACACA A-133911.1  49csascsasasdTs(5MdC)sdAsdTsdTs(5MdC)s(5MdC)sdAsdGsdTsgsasasusg  407 145CAUUCACUGGAAUGAUUGUG 241 CACAAUCAUUCCAGUGAAUG A-133912.1  50ususcsuscs(5MdC)sdTsdGsdGs(5MdC)s(5MdC)s(5MdC)sdAs(5MdC)sdAsasuscsasu 418 146 AUGAUUGUGGGCCAGGAGAA 242 UUCUCCUGGCCCACAAUCAU A-133913.1  51gsususususdGsdAsdTsdAsdAsdGsdGsdTsdTsdTscsuscscsu  431 147AGGAGAAACCUUAUCAAAAC 243 GUUUUGAUAAGGUUUCUCCU A-133914.1  52usasgscsasdTsdTs(5MdC)sdTsdTsdGsdTsdTsdTsdTsgsasusasa  441 148UUAUCAAAACAAGAAUGCUA 244 UAGCAUUCUUGUUUUGAUAA A-133915.1  53cscsasususdAsdTsdTsdGsdAsdTsdAsdGs(5MdC)sdAsususcsusu  451 149AAGAAUGCUAUCAAUAAUGG 245 CCAUUAUUGAUAGCAUUCUU A-133916.1  54gsasusasusdTs(5MdC)sdGsdAsdAs(5MdC)sdAs(5MdC)s(5MdC)sdAsususasusu  463150 AAUAAUGGUGUUCGAAUAUC 246 GAUAUUCGAACACCAUUAUU A-133917.1  55asasascscsdTs(5MdC)sdTsdTs(5MdC)s(5MdC)sdAsdGsdAsdTsasususcsg  475 151CGAAUAUCUGGAAGAGGUUU 247 AAACCUCUUCCAGAUAUUCG A-133918.1  56ususususgsdAs(5MdC)sdAs(5MdC)sdAsdAsdAsdAsdAs(5MdC)scsuscsusu  486 152AAGAGGUUUUUGUGUCAAAA 248 UUUUGACACAAAAACCUCUU A-133919.1  57usgsusasgsdAsdAs(5MdC)sdAsdTsdTsdTsdTsdGsdAscsascsasa  495 153UUGUGUCAAAAUGUUCUACA 249 UGUAGAACAUUUUGACACAA A-133920.1  58ususasasusdTsdGsdGs(5MdC)sdTsdTsdAsdAsdTsdGsusasgsasa  508 154UUCUACAUUAAGCCAAUUAA 250 UUAAUUGGCUUAAUGUAGAA A-133921.1  59cscsusususdAsdTsdAs(5MdC)sdTsdTsdAsdAsdTsdTsgsgscsusu  517 155AAGCCAAUUAAGUAUAAAGG 251 CCUUUAUACUUAAUUGGCUU A-133922.1  60ususcsususdAsdAsdTsdAsdGsdGsdAs(5MdC)s(5MdC)sdTsususasusa  529 156UAUAAAGGUCCUAUUAAGAA 252 UUCUUAAUAGGACCUUUAUA A-133923.1  61asgsusususdTsdTs(5MdC)sdTs(5MdC)s(5MdC)s(5MdC)sdTsdTs(5MdC)sususasasu 541 157 AUUAAGAAGGGAGAAAAACU 253 AGUUUUUCUCCCUUCUUAAU A-133924.1  62asusasgsasdGsdTsdTs(5MdC)s(5MdC)sdAsdAsdGsdTsdTsusususcsu  552 158AGAAAAACUUGGAACUCUAU 254 AUAGAGUUCCAAGUUUUUCU A-133925.1  63gscsasasgsdGsdGs(5MdC)sdAsdAsdTsdAsdGsdAsdGsususcscsa  561 159UGGAACUCUAUUGCCCUUGC 255 GCAAGGGCAAUAGAGUUCCA A-133926.1  64asusasasas(5MdC)sdTsdTsdTs(5MdC)sdTsdGs(5MdC)sdAsdAsgsgsgscsa  572 160UGCCCUUGCAGAAAGUUUAU 256 AUAAACUUUCUGCAAGGGCA A-133927.1  65asususgsusdAsdTsdGs(5MdC)s(5MdC)sdAsdGsdGsdAsdTsasasascsu  585 161AGUUUAUCCUGGCAUACAAU 257 AUUGUAUGCCAGGAUAAACU A-133928.1  66gscsascsasdTsdGs(5MdC)sdGsdAsdTsdTsdGsdTsdAsusgscscsa  594 162UGGCAUACAAUCGCAUGUGC 258 GCACAUGCGAUUGUAUGCCA A-133929.1  67asgsusususdTs(5MdC)sdAsdAsdTsdGsdTsdGs(5MdC)sdAscsasusgsc  606 163GCAUGUGCACAUUGAAAACU 259 AGUUUUCAAUGUGCACAUGC A-133930.1  68ascsuscsgsdAsdGsdTs(5MdC)sdAs(5MdC)sdAsdGsdTsdTsususcsasa  617 164UUGAAAACUGUGACUCGAGU 260 ACUCGAGUCACAGUUUUCAA A-133931.1  69csasgsusasdGsdGsdGsdTs(5MdC)sdAs(5MdC)sdTs(5MdC)sdGsasgsuscsa  627 165UGACUCGAGUGACCCUACUG 261 CAGUAGGGUCACUCGAGUCA A-133932.1  70usususascsdAsdGsdGsdTsdAsdTsdGs(5MdC)sdAsdGsusasgsgsg  639 166CCCUACUGCAUACCUGUAAA 262 UUUACAGGUAUGCAGUAGGG A-133933.1  71usgsgscscsdTsdTs(5MdC)sdGsdAsdTsdTsdTsdAs(5MdC)sasgsgsusa  649 167UACCUGUAAAUCGAAGGCCA 263 UGGCCUUCGAUUUACAGGUA A-133934.1  72asasgsasus(5MdC)sdTsdGsdAs(5MdC)s(5MdC)sdAsdTsdTsdGsgscscsusu  662 168AAGGCCAAUGGUCAGAUCUU 264 AAGAUCUGACCAUUGGCCUU A-133935.1  73usususususdAsdTsdTsdTsdTsdGsdAsdAsdGsdAsuscsusgsa  673 169UCAGAUCUUCAAAAUAAAAA 265 UUUUUAUUUUGAAGAUCUGA A-133936.1  74ususasasgsdAsdTsdGsdAs(5MdC)sdTsdTsdTsdTsdTsasusususu  683 170AAAAUAAAAAGUCAUCUUAA 266 UUAAGAUGACUUUUUAUUUU A-133937.1  75csasuscscsdAsdGsdGsdTsdTsdTsdTsdTsdAsdAsgsasusgsa  694 171UCAUCUUAAAAACCUGGAUG 267 CAUCCAGGUUUUUAAGAUGA A-133938.1  76gsasasgsgsdGsdTsdAsdTsdGs(5MdC)sdAsdTs(5MdC)s(5MdC)sasgsgsusu  704 172AACCUGGAUGCAUACCCUUC 268 GAAGGGUAUGCAUCCAGGUU A-133939.1  77asasususus(5MdC)sdTsdTsdGsdAsdAsdGsdAsdGsdAsasgsgsgsu  717 173ACCCUUCUCUUCAAGAAAUU 269 AAUUUCUUGAAGAGAAGGGU A-133940.1  78gsusgsasas(5MdC)sdAs(5MdC)sdAsdAsdAsdTsdTsdTs(5MdC)sususgsasa  726 174UUCAAGAAAUUUGUGUUCAC 270 GUGAACACAAAUUUCUUGAA A-133941.1  79csasusususdTsdTs(5MdC)s(5MdC)sdTsdTsdTsdGsdTsdGsasascsasc  738 175GUGUUCACAAAGGAAAAAUG 271 CAUUUUUCCUUUGUGAACAC A-133942.1  80csasuscscs(5MdC)sdTsdTs(5MdC)sdAsdTsdGs(5MdC)sdAsdTsususususc  750 176GAAAAAUGCAUGAAGGGAUG 272 CAUCCCUUCAUGCAUUUUUC A-133943.1  81asusgsgsgsdGsdTsdAsdTs(5MdC)s(5MdC)sdAsdTs(5MdC)s(5MdC)scsususcsa  760177 UGAAGGGAUGGAUACCCCAU 273 AUGGGGUAUCCAUCCCUUCA A-133944.1  82asusgsuscsdAsdTsdGsdGsdAsdAsdAsdAsdTsdGsgsgsgsusa  772 178UACCCCAUUUUCCAUGACAU 274 AUGUCAUGGAAAAUGGGGUA A-133945.1  83gsusasasusdAsdAsdTs(5MdC)sdAsdTsdGsdTs(5MdC)sdAsusgsgsasa  781 179UUCCAUGACAUGAUUAUUAC 275 GUAAUAAUCAUGUCAUGGAA A-133946.1  84asgsgscsasdTsdGs(5MdC)sdAsdAsdTsdGsdTsdGsdTsasasusasa  794 180UUAUUACACAUUGCAUGCCU 276 AGGCAUGCAAUGUGUAAUAA A-133947.1  85gsususususdGsdAsdTsdAs(5MdC)sdAsdGsdGs(5MdC)sdAsusgscsasa  804 181UUGCAUGCCUGUAUCAAAAC 277 GUUUUGAUACAGGCAUGCAA A-133948.1  86gsusascsgsdTsdGsdAsdGsdAsdTsdGsdTsdTsdTsusgsasusa  815 182UAUCAAAACAUCUCACGUAC 278 GUACGUGAGAUGUUUUGAUA A-133949.1  87usasusgsusdTsdTsdAsdTsdGsdAsdGsdGsdTsdAscsgsusgsa  827 183UCACGUACCUCAUAAACAUA 279 UAUGUUUAUGAGGUACGUGA A-133950.1  88csasusasgsdGsdTsdGsdTsdAsdTsdAsdTsdGsdTsususasusg  837 184CAUAAACAUAUACACCUAUG 280 CAUAGGUGUAUAUGUUUAUG A-133951.1  89usususgsusdGsdGsdGsdTsdAs(5MdC)sdAsdTsdAsdGsgsusgsusa  847 185UACACCUAUGUACCCACAAA 281 UUUGUGGGUACAUAGGUGUA A-133952.1  90asususasasdAsdAsdAsdAsdTsdTsdTsdTsdTsdGsusgsgsgsu  858 186ACCCACAAAAAUUUUUUAAU 282 AUUAAAAAAUUUUUGUGGGU A-133953.1  91ususcscsusdTsdTsdTsdTsdTsdTsdAsdAsdTsdTsasasasasa  870 187UUUUUAAUUAAAAAAAGGAA 283 UUCCUUUUUUUAAUUAAAAA A-133954.1  92asasascsus(5MdC)sdAsdAsdAsdTsdTsdTs(5MdC)s(5MdC)sdTsususususu  880 188AAAAAAGGAAAUUUGAGUUU 284 AAACUCAAAUUUCCUUUUUU A-133955.1  93usgsususus(5MdC)sdTsdAsdTsdTsdTsdAsdAsdAs(5MdC)suscsasasa  891 189UUUGAGUUUAAAUAGAAACA 285 UGUUUCUAUUUAAACUCAAA A-133956.1  94csususgscsdAsdTsdTsdTsdAsdTs(5MdC)sdAsdTsdGsusususcsu  904 190AGAAACAUGAUAAAUGCAAG 286 CUUGCAUUUAUCAUGUUUCU A-133957.1  95asusgsususdTsdTs(5MdC)sdTsdTsdTs(5MdC)sdTsdTsdGscsasususu  915 191AAAUGCAAGAAAGAAAACAU 287 AUGUUUUCUUUCUUGCAUUU A-133958.1  96asasasasus(5MdC)sdAsdAsdAsdAsdTsdGsdTsdTsdTsuscsususu  924 192AAAGAAAACAUUUUGAUUUU 288 AAAAUCAAAAUGUUUUCUUU A-133959.1  97gsascsasasdTsdGsdAsdGsdTsdTsdAsdAsdAsdAsuscsasasa  935 193UUUGAUUUUAACUCAUUGUC 289 GACAAUGAGUUAAAAUCAAA A-133960.1  98gsasascsasdTs(5MdC)sdAsdGsdAsdGsdTsdGsdAs(5MdC)sasasusgsa  947 194UCAUUGUCACUCUGAUGUUC 290 GAACAUCAGAGUGACAAUGA A-133961.1   99csasgsusus(5MdC)sdAs(5MdC)sdAsdTsdGsdAsdAs(5MdC)sdAsuscsasgsa  957 195UCUGAUGUUCAUGUGAACUG 291 CAGUUCACAUGAACAUCAGA A-133962.1 100asgscscscsdGsdAsdAsdGs(5MdC)sdAsdAs(5MdC)s(5MdC)sdAsgsususcsa  970 196UGAACUGGUUGCUUCGGGCU 292 AGCCCGAAGCAACCAGUUCA A-133963.1 101csasgsasus(5MdC)sdAsdAsdAsdGsdAsdGs(5MdC)s(5MdC)s(5MdC)sgsasasgsc  980197 GCUUCGGGCUCUUUGAUCUG 293 CAGAUCAAAGAGCCCGAAGC A-133964.1 102uscscsasusdAsdGsdGsdTsdGsdAs(5MdC)sdAsdGsdAsuscsasasa  991 198UUUGAUCUGUCACCUAUGGA 294 UCCAUAGGUGACAGAUCAAA A-133965.1 103ascscsascsdTs(5MdC)sdAsdGsdAsdTsdTs(5MdC)s(5MdC)sdAsusasgsgsu 1002 199ACCUAUGGAAUCUGAGUGGU 295 ACCACUCAGAUUCCAUAGGU A-133966.1 104asasasasasdAsdTsdAsdAsdAsdAs(5MdC)s(5MdC)sdAs(5MdC)suscsasgsa 1012 200UCUGAGUGGUUUUAUUUUUU 296 AAAAAAUAAAACCACUCAGA A-133967.1 105csusgsasgsdAsdAsdAsdTs(5MdC)sdTsdAsdAsdAsdAsasasusasa 1023 201UUAUUUUUUAGAUUUCUCAG 297 CUGAGAAAUCUAAAAAAUAA A-133968.1 106usasgsasus(5MdC)sdTsdTsdTsdGsdGsdGsdAs(5MdC)sdTsgsasgsasa 1036 202UUCUCAGUCCCAAAGAUCUA 298 UAGAUCUUUGGGACUGAGAA A-133969.1 107ususasususdTsdAsdTs(5MdC)sdTsdTsdAsdGsdAsdTscsusususg 1046 203CAAAGAUCUAAGAUAAAUAA 299 UUAUUUAUCUUAGAUCUUUG A-133970.1 108gsususcsus(5MdC)sdTsdTsdGsdTsdTsdTsdAsdTsdTsusasuscsu 1056 204AGAUAAAUAAACAAGAGAAC 300 GUUCUCUUGUUUAUUUAUCU

TABLE 4 Mean % inhibition relative to mock transduced sd % A-133928.115.31 5.73 A-133930.1 16.5 5.24 A-133931.1 17.83 2.85 A-133932.1 17.983.05 A-133901.1 18.55 3.79 A-133933.1 19.95 6.21 A-133934.1 20.18 4.28A-133929.1 22.58 11.57 A-133902.1 23.6 2.72 A-133903.1 27.68 4.2A-133909.1 29 13.23 A-133907.1 29.45 9.49 A-133896.1 30.34 5.72A-133899.1 30.56 2.3 A-133891.1 31.51 6.9 A-133906.1 32.09 5.12A-133895.1 32.14 10.8 A-133927.1 33.65 8.46 A-133912.1 34.35 3.53A-133882.1 35.27 3.69 A-133892.1 35.44 1.11 A-133897.1 36.39 2.3A-133925.1 37.18 10.27 A-133880.1 37.43 3.74 A-133962.1 38.35 5.73A-133946.1 38.38 12.31 A-133879.1 38.76 7.22 A-133947.1 38.86 12.52A-133905.1 38.97 3.73 A-133938.1 39.54 10.71 A-133951.1 39.76 8.7A-133900.1 40.16 8.73 A-133965.1 40.35 6.63 A-133894.1 40.88 6.57A-133926.1 41.56 3.95 A-133898.1 42.14 4.02 A-133910.1 42.72 5.48A-133940.1 42.82 11.53 A-133893.1 43.33 8.19 A-133881.1 43.66 3.67A-133884.1 44.45 9.28 A-133918.1 45.11 13.42 A-133924.1 45.15 3.19A-133911.1 45.38 13.17 A-133949.1 45.51 3.91 A-133908.1 45.66 6.29A-133921.1 47.64 11.3 A-133883.1 47.75 2.72 A-133964.1 48.13 3.55A-133948.1 48.75 13.64 A-133952.1 49.2 6.98 A-133919.1 49.46 9.18A-133922.1 50.2 8.04 A-133904.1 51.11 3.64 A-133939.1 52.21 11.03A-133960.1 52.59 8.76 A-133877.1 53.67 14.22 A-133914.1 54.04 14.19A-133888.1 54.1 2.28 A-133917.1 54.86 7.77 A-133889.1 55.19 18.94A-133963.1 55.73 14.28 A-133961.1 56.33 5.71 A-133943.1 56.72 11.3A-133937.1 57.82 8.37 A-133916.1 59.71 11.95 A-133923.1 60.53 15.21A-133935.1 62.15 14.18 A-133945.1 62.37 10.27 A-133878.1 62.8 20.59A-133876.1 62.81 29 A-133936.1 63.23 12.85 A-133953.1 63.82 9.99A-133955.1 64.82 8.41 A-133956.1 65.13 5.53 A-133942.1 66.39 12.33A-133887.1 67.61 11.09 A-133950.1 68.07 17.64 A-133954.1 70.09 7.7A-133966.1 70.84 11.12 A-133890.1 71.79 11.09 A-133957.1 72.28 13.35A-133875.1 73.95 25.21 A-133968.1 75.3 23.16 A-133915.1 76.02 24.82A-133913.1 76.42 13.21 A-133959.1 77.11 12.43 A-133920.1 84.58 5.42A-133941.1 85.16 26.35 A-133967.1 86.78 29.49 A-133970.1 92.06 22.55A-133885.1 96.21 26.94 A-133958.1 106.04 11.27 A-133944.1 111.1 16.85A-133969.1 112.27 29.54 A-133886.1 114.84 26.13

SEQUENCES SEQ ID NO: 1>gi|59806344|ref|NM_002302.2| Homo sapiens leukocyte cell-derived chemotaxi2 (LECT2), mRNAAAATCAAATAGCTATCCATGGAATATTAGAACTTGACTTGCTCCATCCTCTTAAACTTTTTGTGTCTCACACTAAAGAAATGAGAGATGCAGAATTCTAAGGCTAAATAGCTAGGAAGTATTCATTCAAACTTGAATATTCTTCAAAGAGAGTGTGGGGGCAACTCTAATCAGAGGAAGAAACTAAAGGAAGTAAAACCAGATGTTTTCCACCAAAGCCCTCCTTTTGGCTGGTCTGATTTCTACCGCACTGGCAGGGCCATGGGCTAATATATGTGCTGGCAAGTCTTCCAATGAGATCCGGACGTGTGACCGCCATGGCTGTGGACAGTACTCTGCTCAAAGAAGTCAGAGGCCTCACCAGGGTGTGGACATCTTGTGCTCTGCTGGATCTACTGTGTACGCACCATTCACTGGAATGATTGTGGGCCAGGAGAAACCTTATCAAAACAAGAATGCTATCAATAATGGTGTTCGAATATCTGGAAGAGGTTTTTGTGTCAAAATGTTCTACATTAAGCCAATTAAGTATAAAGGTCCTATTAAGAAGGGAGAAAAACTTGGAACTCTATTGCCCTTGCAGAAAGTTTATCCTGGCATACAATCGCATGTGCACATTGAAAACTGTGACTCGAGTGACCCTACTGCATACCTGTAAATCGAAGGCCAATGGTCAGATCTTCAAAATAAAAAGTCATCTTAAAAACCTGGATGCATACCCTTCTCTTCAAGAAATTTGTGTTCACAAAGGAAAAATGCATGAAGGGATGGATACCCCATTTTCCATGACATGATTATTACACATTGCATGCCTGTATCAAAACATCTCACGTACCTCATAAACATATACACCTATGTACCCACAAAAATTTTTTAATTAAAAAAAGGAAATTTGAGTTTAAATAGAAACATGATAAATGCAAGAAAGAAAACATTTTGATTTTAACTCATTGTCACTCTGATGTTCATGTGAACTGGTTGCTTCGGGCTCTTTGATCTGTCACCTATGGAATCTGAGTGGTTTTATTTTTTAGATTTCTCAGTCCCAAAGATCTAAGATAAATAAACAAGAGAACTT SEQ ID NO: 2>gi|380692339|ref|NM_001194884.2| Macaca mulatta leukocyte cell-derivedchemotaxin 2 (LECT2), mRNATTAGAACTTGACTTGCTCCATACTCTTATATTTTTTGTGTCTCACACTAAAGAAATGAAAGATGCATGATTCTAAGGCTAAATAGCTAGGAAGTATTCATTCAAACTTGAATATTCTTCAAAGAGAGTGTGGGGGCAACTCTAATCGGAGGAAGAAACATAAAGGAAGTAAACCCAGATGTTTTCCACCAAAGTCCTCCTTTTGGCTGGTCTGATTTCTACTGCACTGGCAGGGCCATGGGCTAATATATGTGCTGGCAAGTCTTCCAACGAGATCCGGACGTGTGACCGCCATGGCTGTGGACAGTACTCTGCTCAAAGAAGTCAGAGGCCTCACCAGGGTGTGGACATCTTGTGCTCTGCTGGATCTACTGTGTATGCACCATTCACTGGAATGATTGTGGGCCAGGAGAAACCTTATCAAAACAAAAATGCTATCAATAATGGTGTTCGAATATCTGGAAGAGGTTTTTGTGTCAAAATGTTCTACATTAAGCCAATTAAGTATAAAGGTCCTGTTAAGAAGGGAGAAAAACTTGGAACTCTATTGCCCTTGCAGAAAGTTTATCCTGGCATACAGTCGCACGTGCACATTGAAAACTGTGACTTGAGTGACCCTACTGCATACCTGTAAATGGAAGGCCAATGGTCAGATCTTCAAAATAAAAAGTCATCTTAAAAACCTGGATGCATACCCCGCTCTTCAAGAAATTTGTATTCACAAAGGAAAAATGCATGAAGGGATGGATACTCCATTTTCCAGGACTTGGTTATTATACATTGCGTGCTTGTATCAAAAAATCTCACGTACCTCATATATGTATACACTTGTGTACCCACAAAATTTTTT SEQ ID NO: 3>gi|255760038|ref|NM_010702.2| Mus musculus leukocyte cell-derived chemotaxin2 (Lect2), mRNAGGATCAGACAGCTCTCCACGGAATGTTACAGTTTAACCCGACTCATCTTGCTTTCTGTGTCTCTTACTAGATCTCGGAGAGACATAAATTACCAGAGAAGATTCACTCATTCCGGGGGTGACCCTGTGTGCATGGAGCAGGTCTAACTAGAGGAAGAAACAAGAAGCAGAACCTAGATGATTCCCACAACAATCCTCATTTCAGCTGCTTTGCTTTCCTCTGCCCTAGCAGGACCATGGGCTAACATATGTGCCAGCAAATCTTCCAACGAGATCCGGACGTGTGACAGCTATGGCTGTGGACAGTACTCTGCTCAAAGAACCCAAAGGCATCACCCAGGTGTGGACGTCCTGTGCTCGGATGGATCTGTGGTGTATGCACCATTCACTGGGAAGATAGTGGGCCAGGAGAAACCCTATAGAAACAAAAATGCCATCAATGATGGCATTCGACTGTCTGGAAGAGGTTTTTGTGTCAAAATTTTCTACATTAAGCCAATTAAGTATAAAGGTTCTATCAAAAAGGGGGAGAAGCTGGGCACCTTGCTGCCCCTGCAGAAAGTTTACCCGGGCATCCAGTCGCATGTACACGTTGAAAACTGCGACTCCAGTGACCCCACAGCATACCTGTAAGCAGAGACAAAGGCCAGATCTTCTAAATTCAAAGCCATCTCAGAAACTGGGACATGCCCTGCTCCCGAAGAAACGTGCATTCTAACAGAAACATAAATCTGTGTAAACACTCACAACCCTGATTCCAAGTCATCACCGCTCTGACGGGTGGGCTCTGGTCGGCCTGCCACCTCAAGGGCCAGGGAGTTTGACATTTTCGATTTTTAGGTTCTGGTGACTGAGATAAATGAATGACCTCTCATGTGAAAAAAAAAAAA SEQ ID NO: 4>gi|157817277|ref|NM_001108405.1| Rattus norvegicus leukocyte cell-derivedchemotaxin 2 (Lect2), mRNATGACTCATTTTGTTTTCCGTGTCTCGTACTAAACCTCGGAGAAACACGGGATTCCAAAGCTTAATTACCAGAGAAGGTGGACTCAGTCGTGGACTGTGTGTGCATGGGAGCAATTCTAACCAGAGGAAGAAACGGGAAGCAGAATCCAGATGTTTCCCACAAGAATCCTCATGTCAGCTGCTTTGATTTCCACTGCCCTGGCAGGACCATGGGCTAACATATGTGCCAGCAGATCTTCCAACGAGATCCGGACATGTGACAGCTATGGCTGTGGACAGTACTCTACTCAAAGAACCCAAAGGCATCATCCAGGTGTGGACGTCCTGTGCTCAGATGGATCTGTGGTGTATGCGCCATTCACGGGGAAGATAGTGGGCCAGGAGAAACCCTATAGAAACAAAAACGCCATCAATGATGGCGTCCGACTGTCGGGAAGAGGTTTCTGCGTCAAAATTTTCTACATTAAGCCAATTAAGTATAAGGGCTCTATCAGAAAGGGAGAGAAGCTGGGCACCTTGCTGCCCCTGCAGAAAGTTTACCCTGGCATACAGTCGCATGTACACATTGAAAACTGTGACTCCAGTGATCCCACGGCATACCTGTAAGCAGAGACAGAGGCCAGATCTTCTCAAATTCCCAGAAACTGGAACACGTCCTGCTCCTGAAGAAACGTGCATTCTAACAGACGTGCCATCAGTGTGGACACAATTCTGATTCCGACTCAGCGTCACTCTGATGGGCGGGCTCTGATCTGCCTGCTGCCTGGAGGGCCAGGGAGTTTTATGTTTCCGATTTTTAAGTTCTGGGGACTGAAATTAAASEQ ID NO: 5 Reverse Complement of SEQ ID NO: 1AAGTTCTCTTGTTTATTTATCTTAGATCTTTGGGACTGAGAAATCTAAAAAATAAAACCACTCAGATTCCATAGGTGACAGATCAAAGAGCCCGAAGCAACCAGTTCACATGAACATCAGAGTGACAATGAGTTAAAATCAAAATGTTTTCTTTCTTGCATTTATCATGTTTCTATTTAAACTCAAATTTCCTTTTTTTAATTAAAAAATTTTTGTGGGTACATAGGTGTATATGTTTATGAGGTACGTGAGATGTTTTGATACAGGCATGCAATGTGTAATAATCATGTCATGGAAAATGGGGTATCCATCCCTTCATGCATTTTTCCTTTGTGAACACAAATTTCTTGAAGAGAAGGGTATGCATCCAGGTTTTTAAGATGACTTTTTATTTTGAAGATCTGACCATTGGCCTTCGATTTACAGGTATGCAGTAGGGTCACTCGAGTCACAGTTTTCAATGTGCACATGCGATTGTATGCCAGGATAAACTTTCTGCAAGGGCAATAGAGTTCCAAGTTTTTCTCCCTTCTTAATAGGACCTTTATACTTAATTGGCTTAATGTAGAACATTTTGACACAAAAACCTCTTCCAGATATTCGAACACCATTATTGATAGCATTCTTGTTTTGATAAGGTTTCTCCTGGCCCACAATCATTCCAGTGAATGGTGCGTACACAGTAGATCCAGCAGAGCACAAGATGTCCACACCCTGGTGAGGCCTCTGACTTCTTTGAGCAGAGTACTGTCCACAGCCATGGCGGTCACACGTCCGGATCTCATTGGAAGACTTGCCAGCACATATATTAGCCCATGGCCCTGCCAGTGCGGTAGAAATCAGACCAGCCAAAAGGAGGGCTTTGGTGGAAAACATCTGGTTTTACTTCCTTTAGTTTCTTCCTCTGATTAGAGTTGCCCCCACACTCTCTTTGAAGAATATTCAAGTTTGAATGAATACTTCCTAGCTATTTAGCCTTAGAATTCTGCATCTCTCATTTCTTTAGTGTGAGACACAAAAAGTTTAAGAGGATGGAGCAAGTCAAGTTCTAATATTCCATGGATAGCTATTTGATTTSEQ ID NO: 6 Reverse Complement of SEQ ID NO: 2AAAAAATTTTGTGGGTACACAAGTGTATACATATATGAGGTACGTGAGATTTTTTGATACAAGCACGCAATGTATAATAACCAAGTCCTGGAAAATGGAGTATCCATCCCTTCATGCATTTTTCCTTTGTGAATACAAATTTCTTGAAGAGCGGGGTATGCATCCAGGTTTTTAAGATGACTTTTTATTTTGAAGATCTGACCATTGGCCTTCCATTTACAGGTATGCAGTAGGGTCACTCAAGTCACAGTTTTCAATGTGCACGTGCGACTGTATGCCAGGATAAACTTTCTGCAAGGGCAATAGAGTTCCAAGTTTTTCTCCCTTCTTAACAGGACCTTTATACTTAATTGGCTTAATGTAGAACATTTTGACACAAAAACCTCTTCCAGATATTCGAACACCATTATTGATAGCATTTTTGTTTTGATAAGGTTTCTCCTGGCCCACAATCATTCCAGTGAATGGTGCATACACAGTAGATCCAGCAGAGCACAAGATGTCCACACCCTGGTGAGGCCTCTGACTTCTTTGAGCAGAGTACTGTCCACAGCCATGGCGGTCACACGTCCGGATCTCGTTGGAAGACTTGCCAGCACATATATTAGCCCATGGCCCTGCCAGTGCAGTAGAAATCAGACCAGCCAAAAGGAGGACTTTGGTGGAAAACATCTGGGTTTACTTCCTTTATGTTTCTTCCTCCGATTAGAGTTGCCCCCACACTCTCTTTGAAGAATATTCAAGTTTGAATGAATACTTCCTAGCTATTTAGCCTTAGAATCATGCATCTTTCATTTCTTTAGTGTGAGACACAAAAAATATAAGAGTATGGAGCAAGTCAAGTTCTAA SEQ ID NO: 7 Reverse Complement of SEQ ID NO: 3TTTTTTTTTTTTCACATGAGAGGTCATTCATTTATCTCAGTCACCAGAACCTAAAAATCGAAAATGTCAAACTCCCTGGCCCTTGAGGTGGCAGGCCGACCAGAGCCCACCCGTCAGAGCGGTGATGACTTGGAATCAGGGTTGTGAGTGTTTACACAGATTTATGTTTCTGTTAGAATGCACGTTTCTTCGGGAGCAGGGCATGTCCCAGTTTCTGAGATGGCTTTGAATTTAGAAGATCTGGCCTTTGTCTCTGCTTACAGGTATGCTGTGGGGTCACTGGAGTCGCAGTTTTCAACGTGTACATGCGACTGGATGCCCGGGTAAACTTTCTGCAGGGGCAGCAAGGTGCCCAGCTTCTCCCCCTTTTTGATAGAACCTTTATACTTAATTGGCTTAATGTAGAAAATTTTGACACAAAAACCTCTTCCAGACAGTCGAATGCCATCATTGATGGCATTTTTGTTTCTATAGGGTTTCTCCTGGCCCACTATCTTCCCAGTGAATGGTGCATACACCACAGATCCATCCGAGCACAGGACGTCCACACCTGGGTGATGCCTTTGGGTTCTTTGAGCAGAGTACTGTCCACAGCCATAGCTGTCACACGTCCGGATCTCGTTGGAAGATTTGCTGGCACATATGTTAGCCCATGGTCCTGCTAGGGCAGAGGAAAGCAAAGCAGCTGAAATGAGGATTGTTGTGGGAATCATCTAGGTTCTGCTTCTTGTTTCTTCCTCTAGTTAGACCTGCTCCATGCACACAGGGTCACCCCCGGAATGAGTGAATCTTCTCTGGTAATTTATGTCTCTCCGAGATCTAGTAAGAGACACAGAAAGCAAGATGAGTCGGGTTAAACTGTAACATTCCGTGGAGAGCTGTCTGATCCSEQ ID NO: 8 Reverse Complement of SEQ ID NO: 4TTTAATTTCAGTCCCCAGAACTTAAAAATCGGAAACATAAAACTCCCTGGCCCTCCAGGCAGCAGGCAGATCAGAGCCCGCCCATCAGAGTGACGCTGAGTCGGAATCAGAATTGTGTCCACACTGATGGCACGTCTGTTAGAATGCACGTTTCTTCAGGAGCAGGACGTGTTCCAGTTTCTGGGAATTTGAGAAGATCTGGCCTCTGTCTCTGCTTACAGGTATGCCGTGGGATCACTGGAGTCACAGTTTTCAATGTGTACATGCGACTGTATGCCAGGGTAAACTTTCTGCAGGGGCAGCAAGGTGCCCAGCTTCTCTCCCTTTCTGATAGAGCCCTTATACTTAATTGGCTTAATGTAGAAAATTTTGACGCAGAAACCTCTTCCCGACAGTCGGACGCCATCATTGATGGCGTTTTTGTTTCTATAGGGTTTCTCCTGGCCCACTATCTTCCCCGTGAATGGCGCATACACCACAGATCCATCTGAGCACAGGACGTCCACACCTGGATGATGCCTTTGGGTTCTTTGAGTAGAGTACTGTCCACAGCCATAGCTGTCACATGTCCGGATCTCGTTGGAAGATCTGCTGGCACATATGTTAGCCCATGGTCCTGCCAGGGCAGTGGAAATCAAAGCAGCTGACATGAGGATTCTTGTGGGAAACATCTGGATTCTGCTTCCCGTTTCTTCCTCTGGTTAGAATTGCTCCCATGCACACACAGTCCACGACTGAGTCCACCTTCTCTGGTAATTAAGCTTTGGAATCCCGTGTTTCTCCGAGGTTTAGTACGAGACACGGAAAACAAAATGAGTCA

1. An antisense polynucleotide agent for inhibiting expression of LECT2,wherein the agent comprises about 4 to about 50 contiguous nucleotides,wherein at least one of the contiguous nucleotides is a modifiednucleotide, and wherein the nucleotide sequence of the agent is about80% complementary over its entire length to the equivalent region of thenucleotide sequence of any one of SEQ ID NOs:1-4.
 2. (canceled)
 3. Anantisense polynucleotide agent for inhibiting expression of LECT2,wherein the agent comprises at least 8 contiguous nucleotides differingby no more than 3 nucleotides from any one of the nucleotide sequenceslisted in Table
 3. 4.-5. (canceled)
 6. The agent of claim 1, which is 10to 40 nucleotides in length, 10 to 30 nucleotides in length, 18 to 30nucleotides in length, 10 to 24 nucleotides in length, 18 to 24nucleotides in length, or 14 or 20 nucleotides in length. 7.-11.(canceled)
 12. The agent of claim 1, wherein the modified nucleotidecomprises a modified sugar moiety selected from the group consisting of:a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxy modified sugarmoiety, a 2′-O-alkyl modified sugar moiety, and a bicyclic sugar moiety,or wherein the modified nucleotide is a 5-methylcytosine or comprises amodified internucleoside linkage. 13.-16. (canceled)
 17. The agent ofclaim 1, comprising a plurality of 2′-deoxynucleotides flanked on eachside by at least one nucleotide having a modified sugar moiety whereinthe agent is a gapmer comprising a gap segment comprised of linked2′-deoxynucleotides positioned between a 5′ and a 3′ wing segment; orwherein the modified sugar moiety is selected from the group consistingof a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxy modifiedsugar moiety, a 2′-O-alkyl modified sugar moiety, and a bicyclic sugarmoiety. 18.-19. (canceled)
 20. The agent of claim 17, wherein the5′-wing segment is 1 to 6 nucleotides, or is, 3, 4, or 5 nucleotides inlength.
 21. The agent of claim 17, wherein the 3′ -wing segment is 1 to6 or is 2, 3, 4, or 5 nucleotides in length.
 22. The agent of claim 17,wherein the gap segment is 5 to 14 or 10 nucleotides in length. 23.-31.(canceled)
 32. An antisense polynucleotide agent for inhibiting LECT2expression, comprising: a gap segment consisting of linkeddeoxynucleotides; a 5′-wing segment consisting of linked nucleotides; a3′-wing segment consisting of linked nucleotides; wherein the gapsegment is positioned between the 5′-wing segment and the 3′-wingsegment and wherein each nucleotide of each wing segment comprises amodified sugar.
 33. The agent of claim 32, wherein the gap segment isten 2′-deoxynucleotides in length and each of the wing segments is two,three, four, or five nucleotides in length; or wherein the modifiedsugar moiety is selected from the group consisting of a2′-O-methoxyethyl modified sugar moiety, a 2′-methoxy modified sugarmoiety, a 2′-O-alkyl modified sugar moiety, and a bicyclic sugar moiety.34.-37. (canceled)
 38. The agent of claim 1, wherein the agent furthercomprises a ligand.
 39. The agent of claim 38, wherein the antisensepolynucleotide agent is conjugated to the ligand at the 3′-terminus,wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative. 40.(canceled)
 41. The agent of claim 39, wherein the ligand is


42. A pharmaceutical composition for inhibiting expression of a LECT2gene comprising the agent of claim
 1. 43.-47. (canceled)
 48. Apharmaceutical composition comprising the agent of claim 1, and a lipidformulation, wherein the lipid formulation comprises an LNP or an MC3.49.-50. (canceled)
 51. A method of inhibiting LECT2 expression in acell, the method comprising: (a) contacting the cell with the agent ofclaim 1; and (b) maintaining the cell produced in step (a) for a timesufficient to obtain antisense inhibition of a LECT2 gene, therebyinhibiting expression of the LECT2 gene in the cell. 52.-54. (canceled)55. A method of treating a subject having a disease or disorder thatwould benefit from reduction in LECT2 expression, the method comprisingadministering to the subject a therapeutically effective amount of theagent of claim 1, thereby treating the subject.
 56. A method ofpreventing at least one symptom in a subject having a disease ordisorder that would benefit from reduction in LECT2 expression, themethod comprising administering to the subject a prophylacticallyeffective amount of the agent of claim 1, thereby preventing at leastone symptom in the subject having a disorder that would benefit fromreduction in LECT2 expression.
 57. (canceled)
 58. The method of claim55, wherein the disorder is a LECT2-associated disease, and wherein theLECT2-associated disease and wherein the LECT2-associated disease ischosen from an amyloidosis, an elevated risk for developing amyloidosis,a rheumatoid arthritis, and an acute liver injury. 59.-60. (canceled)61. (canceled)
 62. The method of claim 55, further comprisingadministering a second therapy, to the subject.
 63. The method of claim62, wherein the second therapy is a therapy that supports kidneyfunction chosen from dialysis, a diuretic, an angiotensin convertingenzyme (ACE) inhibitor or an angiotensin receptor blocker (ARB); atherapy that supports liver function; or is removal of all or part ofthe organs affected by the amyloidosis.
 64. (canceled)
 65. (canceled)66. The method of claim 55, wherein the agent is administered at a doseof about 0.01 mg/kg to about 100 mg/kg, or at a dose of about 0.5 mg/kgto about 10 mg/kg.
 67. (canceled)
 68. The method of claim 66, whereinthe agent is administered to the subject once a week, twice a week, ortwice a month.
 69. (canceled)
 70. (canceled)
 71. The method of claim 55,wherein the agent is administered to the subject subcutaneously.